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A Dominant Mutant Form of the Herpes Simplex Virus ICP8 Protein Decreases Viral Late Gene Transcription
Abstract
The herpes simplex virus (HSV) infected cell protein 8 (ICP8) is required for viral DNA replication and normal viral gene expression. Previous work in our laboratory has shown that ICP8 may play a role in stimulating late gene expression. In V2.6 cells which express the d105 mutant form of ICP8, synthesis of late proteins and accumulation of the late gC mRNA are reduced during HSV infection (Gao, M., and Knipe, D.M., J Virol. 65, 2666-2675, 1991). To determine if the negative effect of d105 ICP8 on the late gene expression was exerted at the transcriptional level, we measured the levels of mRNAs and transcription from three late genes, gC, UL47, and gD, in V2.6 cells and Vero cells infected with the HSV-1 wild-type virus. In infected V2.6 cells, the levels of late gC and UL47 mRNA were 7- to 12-fold lower than those of infected Vero cells under conditions where the levels of viral DNA replication in these two cell types were similar. The transcription levels of these two late genes in infected V2.6 cells were reduced to similar extents (9- to 14-fold). The levels of accumulated mRNA and transcription of the early-late gD gene also showed parallel reductions in infected V2.6 cells (about 6-fold). In contrast, transcription of the beta pol gene was reduced only slightly (about 2-fold) by d105 ICP8. These results demonstrate that the d105 ICP8 inhibits expression of three viral late genes at the transcriptional level, and in general, the effect of d105 ICP8 on viral gene expression appears to correlate with the extent to which expression of the gene is stimulated by viral DNA synthesis.
... It is required for DNA replication as a single-stranded DNA (ssDNA) binding protein and plays a role in the initiation of DNA replication in conjunction with the origin binding protein UL9 (36)(37)(38)(39)(40). It also has a separate role in the initiation of late gene expression (41,42) and is thought to be important for viral recombination (43). Interestingly, ICP8 is also known to contain an RNase H-like domain homologous to that of HIV integrase (35). ...
... Raltegravir specifically downregulates late gene expression, independently of DNA replication. In addition to its known roles in DNA replication, ICP8 also independently stimulates transcription of at least three late genes, the glycoprotein C (gC), glycoprotein D (gD), and UL47 genes (41,42). To determine the effects of raltegravir on FeHV-1 gene expression, we adopted a methodology similar to that used to originally define the effects of ICP8 on late gene expression (41,42). ...
... In addition to its known roles in DNA replication, ICP8 also independently stimulates transcription of at least three late genes, the glycoprotein C (gC), glycoprotein D (gD), and UL47 genes (41,42). To determine the effects of raltegravir on FeHV-1 gene expression, we adopted a methodology similar to that used to originally define the effects of ICP8 on late gene expression (41,42). To this end, cells were infected at a high multiplicity of infection (MOI), treated with raltegravir, and collected at 6 hpi for quantitative reverse transcription (qRT)-PCR analysis. ...
The rise of drug-resistant herpesviruses is a longstanding concern, particularly among immunocompromised patients. Therefore, therapies targeting viral proteins other than the DNA polymerase that may be less likely to lead to drug-resistant viruses are urgently needed. Using FeHV-1, an alphaherpesvirus closely related to HHV-1 that similarly causes ocular herpes in its natural host, we found that the HIV integrase inhibitor raltegravir targets different stages of the virus life cycle beyond DNA replication and that it does so without developing drug resistance under the conditions tested. This shows that the drug could provide a viable strategy for the treatment of herpesvirus infections.
... They are further subdivided into two subgroups , 1 and 2. The 1 genes are expressed at significant levels in the absence of viral DNA replication but are further increased by viral DNA synthesis, whereas 2 proteins are produced only if viral DNA synthesis takes place. The mechanisms involved in the transition from early to late gene expression is not well understood, but the expression of late genes requires one cis-acting factor, viral DNA replication, and at least three trans-acting factors: ICP4 (Dixon and Schaffer, 1980; Knipe et al., 1978; Watson and Clements, 1980), ICP8 (Chen and Knipe, 1996; Gao and Knipe, 1991), and ICP27 (McCarthy et al., 1989; Rice et al., 1989; Uprichard and Knipe, 1996). The IE protein ICP27 is a nuclear phosphoprotein 1 Current address: Watson Clinic, Lakeland, FL 33805. 2 To whom correspondence and reprint requests should be addressed: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. ...
... The probes used for detecting gC and ICP8 gene transcripts have been described previously (Chen and Knipe, 1996; Godowski and Knipe, 1986). They are ss DNA molecules generated by cloning viral DNA fragments into M13 bacteriophage DNA. ...
... The RNA was used in a hybridization reaction with 10–20 g of ss M13 DNA probes bound per slot onto Nytran filters. The conditions for hybridization were a modification (Chen and Knipe, 1996) of those used by Zhang et al. (1987). Hybridization was performed for 40–48 h at 68°C in plastic scintillation vials containing 2 ml of 2 SSC and 5 Denhardt's solution. ...
The herpes simplex virus infected cell protein 27 (ICP27) is required for the expression of certain early viral proteins and for many late proteins during productive infection. Expression of at least one late (γ2) gene, that encoding glycoprotein C, is severely restricted in the absence of functional ICP27. The exact mode of action by which ICP27 induces late gene expression is not known, but the effect is apparent at the mRNA level as demonstrated by Northern blot analysis. To determine whether ICP27 activates late genes via transcriptional or posttranscriptional mechanisms, we initially used nuclear run-on assays to measure transcription of viral genes in Vero cells infected with wild-type (WT) virus or an ICP27 nonsense mutant virus, n504. We observed a 4-fold reduction in the nuclear run-on signal from the coding strand of the gC gene for n504-infected cells compared to that of WT-infected cells. However, interpretation of the results was complicated by the observation of a significant signal from the noncoding strand in these experiments. To obviate the problem of symmetrical transcription, we utilized in vivo RNA pulse-labeling to measure the amount of transcription of viral genes in cells infected with either WT virus or n504 virus. We found a 5- to 10-fold reduction in the transcription of the gC and UL47 genes, two late genes, in cells infected with n504 compared to that in cells infected with WT virus. In contrast, transcription of the ICP8 gene, an early gene, was similar in WT and n504 virus-infected cells. We also examined the stability of the gC and UL47 gene transcripts in n504-infected cells, and we found it to be comparable to that in WT virus-infected cells, further supporting an effect on transcription. Transcription of the gC and UL47 genes by n504 was normal in a cell line that expresses WT ICP27. From these results we conclude that ICP27 is required for transcription of the late gC and UL47 genes during productive infection.
... The d105 ICP8 mutant contains a deletion near its C terminus (residues 1082 to 1169), which leaves the nuclear localization signal intact. Previous reports have demonstrated that d105 ICP8 acts as a dominantnegative repressor of wild-type ICP8 activity (6,15). When expressed in Vero cells, either by transient transfection or in a stably transfected cell line, d105 ICP8 inhibits the replication of wild-type virus by 50-to 100-fold. ...
... To study these effects, we isolated a cell line that stably expresses the d105 ICP8 protein (V2.6 cell line) (15). When V2.6 cells are infected with wild-type HSV-1, there is a fivefold reduction in DNA replication and a 50-to 100-fold reduction in late gene expression (15), which is manifested at the transcriptional level (6). Previous experiments have demonstrated that the 50-to 100-fold repression of late gene expression in V2.6 cells is far greater than what would occur in normal infection with DNA replication levels reduced to 20% of the wild-type level (6,15). ...
... When V2.6 cells are infected with wild-type HSV-1, there is a fivefold reduction in DNA replication and a 50-to 100-fold reduction in late gene expression (15), which is manifested at the transcriptional level (6). Previous experiments have demonstrated that the 50-to 100-fold repression of late gene expression in V2.6 cells is far greater than what would occur in normal infection with DNA replication levels reduced to 20% of the wild-type level (6,15). Here, we demonstrate that the d105 ICP8 protein fails to localize to replication compartments and prevents wild-type ICP8 and the other replication proteins and transcription factors from localizing to prereplicative sites and replication compartments as well. ...
The d105 dominant-negative mutant form of the herpes simplex virus 1 (HSV-1) single-stranded DNA-binding protein, ICP8 (d105 ICP8), inhibits wild-type viral replication, and it blocks both viral DNA replication and late gene transcription, although
to different degrees (M. Gao and D. M. Knipe, J. Virol. 65:2666–2675, 1991; Y. M. Chen and D. M. Knipe, Virology 221:281–290,
1996). We demonstrate here that this protein is also capable of preventing the formation of intranuclear prereplicative sites
and replication compartments during HSV infection. We defined three patterns of ICP8 localization using indirect immunofluorescence
staining of HSV-1-infected cells: large replication compartments, small compartments, and no specific intranuclear localization
of ICP8. Cells that form large replication compartments replicate viral DNA and express late genes. Cells that form small
replication compartments replicate viral DNA but do not express late genes, while cells without viral replication compartments
are incapable of both DNA replication and late gene expression. The d105 ICP8 protein blocks formation of prereplicative sites and large replication compartments in 80% of infected cells and
formation of large replication compartments in the remaining 20% of infected cells. The phenotype ofd105 suggests a correlation between formation of large replication compartments and late gene expression and a role for intranuclear
rearrangement of viral DNA and bound proteins in activation of late gene transcription. Thus, these results provide evidence
for specialized machinery for late gene expression within replication compartments.
... An additional protein needed for late gene transcription is the single-stranded-DNA-binding protein, ICP8, one of the seven viral  proteins necessary and sufficient for viral DNA replication in transfected cells (13,96). ICP8 negatively affects transcription from the parental viral genomes (28)(29)(30) but stimulates late gene transcription from progeny DNA templates (15,27). ...
... ICP8 plays a role in stimulating transcription of late genes, which is independent of viral DNA replication (15,27). Its interaction with Pol II is likely a reflection of that function. ...
Herpes simplex virus 1 (HSV-1) infection causes the shutoff of host gene transcription and the induction of a transcriptional program of viral gene expression. Cellular RNA polymerase II is responsible for transcription of all the viral genes, but several viral proteins stimulate viral gene transcription. ICP4 is required for all delayed-early and late gene transcription, ICP0 stimulates transcription of viral genes, and ICP27 stimulates expression of some early genes and transcription of at least some late viral genes. The early DNA-binding protein, ICP8, also stimulates late gene transcription. We therefore investigated which HSV proteins interact with RNA polymerase II. Using immunoprecipitation and Western blotting methods, we observed the coprecipitation of ICP27 and ICP8 with RNA polymerase II holoenzyme. The association of ICP27 with RNA polymerase II was detectable as early as 3 h postinfection, while ICP8 association became evident by 5 h postinfection, and the association of both was independent of viral DNA synthesis. Infections with ICP27 gene mutant viruses revealed that ICP27 is required for the association of ICP8 with RNA polymerase II, while studies with ICP8 gene deletion mutants showed no apparent role for ICP8 in the association of ICP27 with RNA polymerase II. The association of ICP27 and ICP8 with RNA polymerase II holoenzyme appeared to be independent of nucleic acids. We hypothesize that the interaction of ICP27 with RNA polymerase II holoenzyme reflects its role in stimulating early and late gene expression and/or its role in inhibiting host transcription and that the interaction of ICP8 with RNA polymerase II holoenzyme reflects its role in stimulating late gene transcription.
... When expressed in the context of wild-type viral infection, either via transfection or in the stably expressing cell line V2.6, d105 ICP8 reduces viral replication by 50-to 100-fold (Chen and Knipe, 1996;Gao and Knipe, 1991). The reduced viral replication is due to a 5-fold reduction in viral DNA synthesis and a 50-to 100-fold block in late gene transcription (Chen and Knipe, 1996). ...
... When expressed in the context of wild-type viral infection, either via transfection or in the stably expressing cell line V2.6, d105 ICP8 reduces viral replication by 50-to 100-fold (Chen and Knipe, 1996;Gao and Knipe, 1991). The reduced viral replication is due to a 5-fold reduction in viral DNA synthesis and a 50-to 100-fold block in late gene transcription (Chen and Knipe, 1996). Overexpression of wild-type ICP8 can overcome this block, suggesting that d105 ICP8 acts in a competitive manner (Gao and Knipe, 1991). ...
The herpes simplex virus single-stranded DNA-binding protein, ICP8, localizes initially to structures in the nucleus called prereplicative sites. As replication proceeds, these sites mature into large globular structures called replication compartments. The details of what signals or proteins are involved in the redistribution of viral and cellular proteins within the nucleus between prereplicative sites and replication compartments are poorly understood; however, we showed previously that the dominant-negative d105 ICP8 does not localize to prereplicative sites and prevents the localization of other viral proteins to prereplicative sites (J. Virol. 74 (2000) 10122). Within the residues deleted in d105 (1083 to 1168), we identified a region between amino acid residues 1080 and 1135 that was predicted by computer models to contain two alpha-helices, one with considerable amphipathic nature. We used site-specific and random mutagenesis techniques to identify residues or structures within this region that are required for proper ICP8 localization within the nucleus. Proline substitutions in the predicted helix generated ICP8 molecules that did not localize to prereplicative sites and acted as dominant-negative inhibitors. Other substitutions that altered the charged residues in the predicted alpha-helix to alanine or leucine residues had little or no effect on ICP8 intranuclear localization. The predicted alpha-helix was dispensable for the interaction of ICP8 with the U(L)9 origin-binding protein. We propose that this C-terminal alpha-helix is required for localization of ICP8 to prereplicative sites by binding viral or cellular factors that target or retain ICP8 at specific intranuclear sites.
... The n504 ICP27 nonsense mutant virus, which encodes the Nterminal 504 amino acid residues of ICP27, is defective for L gene (g2) expression, but replicates near wt levels of DNA (Jean et al., 2001;Rice and Knipe, 1990). The d105 ICP8 mutant is a dominant-negative form of ICP8 that lacks residues 1083-1166 (Chen and Knipe, 1996;Gao and Knipe, 1991). Cells expressing d105 ICP8 and infected with wt HSV-1 exhibited markedly reduced mRNA and protein levels of g1 (ICP5, ICP25) and g2 (ICP15, gC, U L 47) genes compared with wt virus-infected cells. ...
... Cells expressing d105 ICP8 and infected with wt HSV-1 exhibited markedly reduced mRNA and protein levels of g1 (ICP5, ICP25) and g2 (ICP15, gC, U L 47) genes compared with wt virus-infected cells. Furthermore, the effects on L gene transcription were independent of viral DNA synthesis (Chen and Knipe, 1996). Given that mutations in these two genes impart similar functional consequences, these data suggest that ICP27 and ICP8 collaborate in the mechanism by which L genes are expressed. ...
Herpes simplex virus 1 (HSV-1) ICP27 and ICP8 proteins have both been implicated in the transcription of late genes and regulation of viral gene expression. We showed previously that ICP27 and ICP8 associate with the RNAP II holoenzyme (Zhou and Knipe, J. Virol. 76, 5893-5904). Here, we demonstrate that ICP27 and ICP8 coprecipitate from lysates of HSV-1-infected HEp2 cells and from lysates of insect cells expressing ICP27 and ICP8, the latter being in the absence of other HSV-1 proteins. By expressing and purifying hexahistidine-tagged ICP8 (His-ICP8) and maltose binding protein (MBP)-tagged ICP27 (MBP-27) proteins and performing in vitro immunoprecipitation and pull-down assays, we also demonstrate that ICP27 and ICP8 coprecipitate in the absence of other viral or cellular proteins. Taken together, these data provide evidence that ICP27 and ICP8 interact directly in vitro and in infected cells. We hypothesize that the ICP27-ICP8 interaction plays a role in the stimulation of late gene transcription.
... In addition to its role in viral DNA amplification, the viral early protein ICP8 -a single stranded DNA-binding proteinis thought to play a role in viral late gene expression. In effect, in cells expressing a dominant negative form of ICP8, both late protein synthesis and the accumulation of late gC mRNA are reduced during HSV-1 infection (Godowski and Knipe, 1986;Gao and Knipe, 1991;Chen and Knipe, 1996). However, it is difficult to conclude since late gene expression is dependent on viral DNA replication. ...
During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that can be divided into three general stages: immediate-early (IE), early (E) and late (L). This expression program is the result of a complex interplay between viral and cellular factors at both the transcriptional and post-transcriptional levels, as well as structural differences within the promoter architecture for each of the three gene classes. Since the cellular enzyme RNA polymerase II (RNAP-II) is responsible for the transcription of herpesvirus genes, most viral promoters contain DNA motifs that are common with those of cellular genes, although promoter complexity decreases from immediate-early to late genes. Immediate-early and early promoters contain numerous cellular and viral cis-regulating sequences upstream of a TATA box, whereas late promoters differ significantly in that they lack cis-acting sequences upstream of the Transcription Start Site (TSS). Moreover, in the case of the β- and γ-herpesviruses, a TATT box motif is frequently found in the position where the consensus TATA box of eukaryotic promoters usually localizes. The mechanisms of transcriptional regulation of the late viral gene promoters appear to be different between α-herpesviruses and the two other herpesvirus subfamilies ( and ). In this review, we will compare the mechanisms of late gene transcriptional regulation between HSV-1, for which the viral IE transcription factors - especially ICP4 - play an essential role, and the two other subfamilies of herpesviruses, with a particular emphasis on EBV, which has recently been found to code for its own specific TATT-binding protein.
... In accord with its ability to destabilize DNA helices (5) and promote Mg-dependent complementary-strand renaturation (16), ICP8 can catalyze homologous pairing and strand transfer (7), and hence it participates in the frequent DNA recombination events. Genetic evidence implies a role for ICP8 in the regulation of viral gene expression (12,26), in agreement with the reported ICP8 affinity for polyriboadenylate (61). ...
ICP8 is the major single-stranded DNA (ssDNA) binding protein of the herpes simplex virus type 1 and is required for the onset and maintenance of viral genomic replication. To identify regions responsible for the cooperative binding to ssDNA, several mutants of ICP8 have been characterized. Total reflection X-ray fluorescence experiments on the constructs confirmed the presence of one zinc atom per molecule. Comparative analysis of the mutants by electrophoretic mobility shift assays was done with oligonucleotides for which the number of bases is approximately that occluded by one protein molecule. The analysis indicated that neither removal of the 60-amino-acid C-terminal region nor Cys254Ser and Cys455Ser mutations qualitatively affect the intrinsic DNA binding ability of ICP8. The C-terminal deletion mutants, however, exhibit a total loss of cooperativity on longer ssDNA stretches. This behavior is only slightly modulated by the two-cysteine substi- tution. Circular dichroism experiments suggest a role for this C-terminal tail in protein stabilization as well as in intermolecular interactions. The results show that the cooperative nature of the ssDNA binding of ICP8 is localized in the 60-residue C-terminal region. Since the anchoring of a C- or N-terminal arm of one protein onto the adjacent one on the DNA strand has been reported for other ssDNA binding proteins, this appears to be the general structural mechanism responsible for the cooperative ssDNA binding by this class of protein. Single-stranded (ssDNA) DNA binding proteins (SSBs) bind preferentially ssDNA in stoichiometric quantities with respect to their substrate, displaying little sequence preference and no associated ATPase activity (11). The binding is typically cooperative, though the level of cooperativity varies widely. Much effort has been spent on elucidation of the structural mechanism accounting for the cooperativity and its functional
... It has also been reported that the DNA binding protein ICP8 is required for L gene expression, but this has been difficult to evaluate independently of its role in DNA replication. However, certain mutants of ICP8 inhibit L gene expression to a far greater extent than they inhibit viral DNA replication, suggesting a more direct role in L gene expression (Chen and Knipe, 1996;McNamee et al., 2000), or that the rate of L gene expression may be dependent on the rate of viral DNA replication as opposed to the absolute amount of DNA that has been synthesised at any one time point. Alternatively, viral protein(s) may have a dual role in DNA replication and L gene activation, similar to the situation described in the replication of phage T4, where proteins required for DNA replication are also required for transcription of phage L genes (Herendeen et al., 1989(Herendeen et al., , 1992Tinker et al., 1994). ...
A significant characteristic of the herpesviruses is that they form latent infections in infected hosts, and can be reactivated to again induce lytic infections by stressors. This thesis deals with an epidemiological investigation of equine herpesvirus infection, particularly gammaherpesvirus infections, in foals and if there was evidence of reactivation of latent virus infections by stressors such as those associated with weaning.
A longitudinal study of EHV infections in young foals and the effect of stressors such as weaning on the prevalence of virus infection was undertaken by the detection of DNA and mRNA of EHV2, EHV5, EHV1 and EHV4 in peripheral blood leukocyte (PBL) and nasal swabs (NS) from 13 mares and 46 foals in 4 stables. EHV2 and EHV5 infections were detected commonly in the study population but infections with the alphaherpesviruses EHV1 and EHV4 were not detected, although lytic infections by the alphaherpesviruses may have been missed due to the frequency of sampling. Age differences in the prevalence of EHV2 and EHV5 infection were detected: the prevalence of EHV2 was higher in young foals than in older foals and adult animals; the prevalence of EHV5 was higher in older foals and adults than in younger foals. The prevalence of EHV2 and EHV5 infection increased in association with weaning, presumably in association with stressors associated with weaning, but was not clearly associated with disease in the weaned animals. It was also observed that EHV5 produced a transient lytic infection in PBL of young foals but tended to produce a persistent lytic infection of PBL in older foals and adults. Persistent lytic EHV5 infections of ¡Ý37 weeks duration were also detected in 2 of 13 adult mares and this has not been reported previously. The persistent lytic infection of PBL was not associated with the detection of virus in NS and the mares with persistent lytic infection of PBL with EHV5 did not transfer the infection to their foals.
To determine if any of the animals examined were latently infected with the alphaherpesviruses, an examination for transcripts of genes 63 and 64 of EHV1 and EHV4, putative latency-associated transcripts (LAT) of EHV, was undertaken. Evidence of these transcripts was detected in PBL and bronchiolar lymph nodes in the absence of transcripts of the structural gB, supporting previous studies indicating that transcripts of genes 63 and 64 may represent LAT. In PBL, EHV1 gene 64 RNA transcripts but not gene 63 transcripts were detected in PBL. In bronchiolar lymph nodes, EHV1 gene 64 (but not gene 63) RNA transcripts were detected. In contrast, EHV4 infection was detected in the trigeminal ganglia only and there was no evidence of EHV4 infection in lymph nodes or PBL. In the trigeminal ganglion, EHV4 gB DNA and gene 63 RNA transcripts were detected. The presence of RNA transcripts of EHV1 gene 64 in PBL and lymph node in the absence of any evidence of the replication of structural proteins suggests PBL and lymph node are sites of EHV1 latent infections. The presence of EHV4 gene 63 transcripts in trigeminal ganglia in the absence of any evidence of replication of structural proteins suggests the trigeminal ganglion is the major site of latency of EHV4.
As a potential means of detecting latency of the gammaherpesvirus EHV2, 4 EHV2 genes ORF74, E4, E8 and E10 were selected as having possible roles during EHV2 latency based on sequence analysis and comparison with gene products identified or postulated as having roles in latency in other gammaherpesviruses. Kinetic transcription of these genes was evaluated in an in vitro time course study using a non neuronal cell line (equine kidney [EK] cells). While the gB and gH genes encoding structural glycoproteins were abundantly transcribed in vitro, the 4 putative EHV2 latency-associated genes were minimally transcribed during lytic infection in EK cells, a result analogous to results obtained for the expression of LATs in other gammaherpesviruses. Attempts to demonstrate transcription products of these genes in PBL or other tissues of horsed presumed to be latently infected with EHV2 (in which gB transcripts had been detected previously) and actively infected with EHV2 (in which gB transcripts had been detected at the time of sampling), were unsuccessful.
... A putative herpes virus recombinase pR1 homologous to RAG-1 with additional N-terminal amino acid sequences in both proteins [17] would also provide amino terminal protein sequences present in RAG-1, and bind to a primordial RAG-2 protein (pR2) co-expressed in somatic cells prior to the origins of the acquired immune system in the sea urchin. As proposed for pR1, the function of amino terminal sequences in the herpes ICP-8 protein are not directly related to DNA binding properties of the protein but rather seem to associate with cellular factors and regulate other viral genes [58][59][60][61][62]. ...
The RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the "RAG transposon".
Novel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a "RAG transposon." A subsequent "arms race" between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex).
A "co-regulatory" model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the "RAG-transposon" hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination.
... (Farrell et al., 1994;McLean et al., 1994;Peeters et al., 1994). However, if the deleted essential gene is an early gene that is required to activate other viral genes, then the number of viral proteins synthesized may be limited and the viral genome may not be able to complete even a single cycle of replication (Chen and Knipe, 1996;Brehm et al., 1997;Da Costa et al., 1997). ...
Whatever strategy is adopted for the development of viral vectors for delivery of veterinary vaccines there are several key points to consider: (1) Will the vectored vaccine give a delivery advantage compared to what's already available? (2) Will the vectored vaccine give a manufacturing advantage compared to what's already available? (3) Will the vectored vaccine provide improved safety compared to what's already available? (5) Will the vectored vaccine increase the duration of immunity compared to what's already available? (6) Will the vectored vaccine be more convenient to store compared to what's already available? (7) Is the vectored vaccine compatible with other vaccines? If there is no other alternative available then the answer to these questions is easy. However, if there are alternative vaccines available then the answers to these questions become very important because the answers will determine whether a vectored vaccine is merely a good laboratory idea or a successful vaccine.
... One early protein with a role in transcriptional regulation is the viral ssDNA binding protein, ICP8. ICP8 is necessary for transcription of late genes beyond its role in DNA replication (71,72). This may involve rearrangement of the viral genome to allow late-specific transcription factors access to the promoters or initiation of a switch to transcription from progeny genomes (73). ...
Herpes simplex virus (HSV) commonly causes human infections in the orofacial region (HSV-1) and in the genital region (HSV-2). Productive viral infection in mucosal epithelial cells may result in clinical symptoms and is followed by a latent infection within sensory neurons. During productive infection a large number of viral gene products are expressed while during latent infection few or no viral proteins are expressed. Reactivation from latency results in recurrent infections and disease at or near the primary site of infection. Understanding the details of the two stages of the HSV life cycle is a particular focus of current research on HSV. The virus interacts with and modifies numerous host cell functions in both epithelial and neuronal cells, and studies of HSV have enhanced our knowledge of many fundamental processes in eukaryotic cells. Ongoing research continues to uncover novel effects of HSV on cells, and a complete understanding of HSV infection during both productive and latent infection should allow the design of new antiviral agents and vaccines and increased knowledge of basic cell and molecular biology. This review article is designed to provide an introduction to HSV biology and key aspects of the infection cycle.
... The HSV-1 mutants TL4 and TL5, which map in the N terminus of ICP8 appears to fully complement the HSV-1 mutant n11SV in which 28 amino acids at the very C terminus have been replaced by the SV40 nuclear localization signal (31). A distinct role for the C terminus of ICP8 is also indicated by the fact that the mutant dl105 with a deletion mapping at the C terminus acts in a dominant-negative way and inhibits viral DNA replication (32). Together these observations argue that ICP8 has at least two distinct roles. ...
The Herpes simplex virus type I origin-binding protein, OBP, is encoded by the UL9 gene. OBP binds the origin of DNA replication, oriS, in a cooperative and sequence-specific manner. OBP is also an ATP-dependent DNA helicase. We have recently shown that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by OBP. OriS* contains a stable hairpin formed by complementary base pairing between box I and box III in oriS. Here we show that OBP, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-base pair double-stranded minimal oriS fragment to oriS* and form an OBP-oriS* complex. The formation of an OBP-oriS* complex requires hydrolysable ATP. We also demonstrate that OBP in the presence of ICP8 and ATP promotes slow but specific and complete unwinding of duplex minimal oriS. The possibility that the OBP-oriS* complex may serve as an assembly site for the herpes virus replisome is discussed.
... ICP8 also has the ability to destabilize doublestranded DNA (Boehmer and Lehman, 1993a) and can promote DNA strand transfer (Bortner et al., 1993) and renaturation of complementary DNA strands (Dutch and Lehman, 1993), which suggests that ICP8 may play a role in HSV genome recombination. In addition to its role in viral DNA synthesis, ICP8 has also been shown to regulate viral gene expression by repressing transcription from the parental genome (Godowski and Knipe, 1983;Godowski and Knipe, 1985;Godowski and Knipe, 1986) and stimulating late gene expression from progeny genomes (Chen and Knipe, 1996;Gao and Knipe, 1991). ...
Herpes simplex virus (HSV) uses intranuclear compartmentalization to concentrate the viral and cellular factors required for the progression of the viral life cycle. Processes as varied as viral DNA replication, late gene expression, and capsid assembly take place within discrete structures within the nucleus called replication compartments. Replication compartments are hypothesized to mature from a few distinct structures, called prereplicative sites, that form adjacent to cellular nuclear matrix-associated ND10 sites. During productive infection, the HSV single-stranded DNA-binding protein ICP8 localizes to replication compartments. To further the understanding of replication compartment maturation, we have constructed and characterized a recombinant HSV-1 strain that expresses an ICP8 molecule with green fluorescent protein (GFP) fused to its C terminus. In transfected Vero cells that were infected with HSV, the ICP8-GFP protein localized to prereplicative sites in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA) or to replication compartments in the absence of PAA. A recombinant HSV-1 strain expressing the ICP8-GFP virus replicated in Vero cells, but the yield was increased by 150-fold in an ICP8-complementing cell line. Using the ICP8-GFP protein as a marker for replication compartments, we show here that these structures start as punctate structures early in infection and grow into large, globular structures that eventually fill the nucleus. Large replication compartments were formed by small structures that either moved through the nucleus to merge with adjacent compartments or remained relatively stationary within the nucleus and grew by accretion and fused with neighboring structures.
... Activation of these cellular signal transduction pathways was also implicated in contributing to the CHX-induced stimulation of the HSV-1 IE promoters for ICP0 and ICP27 as well as the major IE promoter of HCMV (MIEP) when it was placed into the HSV-1 genome (Preston et al., 1998). It is interesting to speculate that these pathways may induce expression of the single-stranded DNA binding protein, as this protein plays a central role in DNA synthesis and, by analogy to its HSV counterpart, may also regulate viral gene expression (Chen and Knipe, 1996; Gao and Knipe, 1991). Experiments to test the regions around each UL57 transcriptional start site for promoter function confirmed that each could serve as a functional promoter in transient assays . ...
The human cytomegalovirus (HCMV) UL57 gene lies adjacent to HCMV oriLyt, from which it is separated by an organizationally conserved, mostly noncoding region that is thought to both regulate UL57 expression and activate oriLyt function. However, the UL57 promoter has not been studied. We determined the 5' ends of UL57 transcripts toward an understanding of the potential relationship between UL57 expression and oriLyt activation. The results presented here identified three distinct 5' ends spread over 800 bp, at nt 90302, 90530, and 91138; use of these sites exhibited differential sensitivity to phosphonoformic acid treatment. Interestingly, a 10-kb UL57 transcript accumulated in cycloheximide-treated infected cells, even though other early transcripts were not detectable. However, the 10-kb transcript did not accumulate in cells treated with the more stringent translation inhibitor anisomycin. Consistent with the notion that the identified 5' ends arise from distinct transcription start sites, the sequences upstream of sites I and II functioned as promoters responsive to HCMV infection in transient assays. However, the origin-proximal promoter region III required downstream sequences for transcriptional activity. Mutation of candidate core promoter elements suggested that promoter III is regulated by an initiator region (Inr) and a downstream promoter element. Finally, a 42-bp sequence containing the candidate Inr activated a minimal oriLyt core construct in transient replication assays. Thus, these studies showed that a large, complex promoter region with novel features controls UL57 expression, and identified a sequence that regulates both UL57 transcription and oriLyt activation.
... Studies in the HSV-1 major DNA-binding protein ICP8 have been linked to multiple functions within the HSV replication cycle. Its ability to modulate activity of the HSV-1 DNA polymerase (Hernandez, 1990), affect genome recombination and processing (Bortner, 1993; Dutch, 1993;), regulate late viral gene expression (Chen, 1996; Gao, 1989;), organize DNA replication enzymes into nuclear replication compartments (Bush, 1991; de Bruyn Kops, 1988) as well as interactions with the helicase-primase complex (Boehmer, 1998; Boehmer, 1993; Crute, 1991; Falkenberg, 1998; Hamatake, 1997; He, 2001; Lee, 1997; Makhov, 1996) provides an insight into potential functional parallels that may be found with its HCMV counterpart. However, it has already been established that like ICP8, UL57 serves as an essential component for transient complementation in HCMV oriLyt-mediated DNA replication (Pari, 1993; Sarisky, 1996). ...
Six of the eleven genes essential for Human cytomegalovirus (HCMV) DNA synthesis have been analyzed for putative structural motifs that may have a significant functional role in DNA replication. The genes studied encode for the DNA polymerase accessory protein (UL44), single-stranded DNA binding protein (UL57), primase-helicase complex (UL70, UL102, and UL105), and the putative initiator protein (UL84). The full-length open reading frames of these genes were highly conserved between ten isolates with amino acid sequence identity of >97% for all genes. Using ScanProsite software from the Expert Protein Analysis System (ExPASy) proteomics server, we have mapped putative motifs throughout these HCMV replication genes. Interesting motifs identified include casein kinase-2 (CKII) phosphorylation sites, a microbodies signal motif in UL57, and an ATP binding site in the putative UL105 helicase. Our investigations have also elucidated motif-rich regions of the UL44 DNA polymerase accessory protein and identified cysteine motifs that have potential implications for UL57 and UL70 primase. Taken together, these findings provide insights to regions of these HCMV replication proteins that are important for post-translation modification, activation, and overall function, and this information can be utilized to target further research into these proteins and advance the development of novel antiviral agents that target these processes.
In this chapter, we present recently acquired information and ideas about transcriptional activation by a sliding-clamp protein. The following topics are touched upon: (1) the biological context of bacteriophage T4 multiplication in which this mechanism operates is briefly reviewed; (2) the activation mechanism is described; (3) a mechanism for coupling selective gene expression to concurrent replication is proposed; (4) information on protein-protein interactions that are required for the transcriptional activation is presented.
The chapter closes with a brief comment on the contrast between physically bound and topologically linked transcriptional activators, and on the completely disproportionate representation of physically bound transcriptional effectors in the catalog of known transcriptional regulation.
Expression of the more than 80 individual genes of herpes simplex virus 1 (HSV-1) takes place in a tightly regulated sequential manner that was first described over 20 years ago. Investigations since that time have focused on understanding the mechanisms that regulate this orderly and efficient expression of viral genes. This review examines recent findings that have shed light on how this process is regulated during productive infection of the cell. Although the story is still not complete, several aspects of HSV gene expression are now clearer as a result of these findings. In particular, several new functions have recently been ascribed to some of the known viral regulatory proteins. The results indicate that the viral gene expression is regulated through transcriptional as well as post-transcriptional mechanisms. In addition, it has become increasingly clear that the virus has evolved specific functions to interact with the host cell in order to divert and redirect critical host functions for its own needs. Understanding the interactions of HSV and the host cell during infection will be essential for a complete understanding of how viral gene expression is regulated. Future challenges in the field will be to develop a complete understanding of the mechanisms that temporally regulate virus gene expression, and to identify and characterize the relevant interactions between the virus and the distinctive cell types normally infected by the virus.
Crystals of a 60-amino-acid C-terminal deletion mutant of the herpes simplex virus 1 single-stranded DNA binding protein, ICP8, have been grown by hanging drop vapor diffusion. The colorless crystals grow as thin plates to a maximum size of approximately 0.3 mm × 0.3 mm × 0.05 mm. The space group is P212121 with unit cell constants a = 101.2 Å, b = 145.8 Å, and c = 162.9 Å. There are one or two molecules of ICP8 per asymmetric unit.
That the expression of late genes is coupled to viral genome replication is well established for all herpesviruses, but the
exact mechanisms of their regulation, especially by viral proteins, are poorly understood. Here, we report the identification
of the Epstein-Barr virus (EBV) early protein BcRF1 as a viral factor crucial for the activation of late gene transcription
following viral DNA replication during the productive cycle. In order to study the function of the BcRF1 protein, we constructed
a recombinant EBV lacking this gene. In HEK293 cells, this recombinant virus underwent normal DNA replication during the productive
cycle but failed to express high levels of late gene transcripts or proteins, resulting in a nonproductive infection. Interestingly,
a TATT motif is present in the promoter of most EBV late genes, at the position of the TATA box. We show here that BcRF1 forms
a complex with the TATT motif and that this interaction is required for activation of late viral gene expression. Moreover,
our results suggest that BcRF1 acts via interaction with other viral proteins.
A replication-defective mutant of herpes simplex virus 2 (HSV-2) was engineered by replacing the ICP8 gene of HSV-2 strain 186 with an ICP8–lacZ fusion gene from the herpes simplex virus 1 (HSV-1) HD-2 mutant strain. The resulting virus, HSV-2 5BlacZ, is defective for growth in Vero cells but is capable of growth in a cell line that expresses HSV-1 ICP8. In Vero cells, the mutant virus is defective for DNA synthesis but is able to express many viral proteins at levels similar to those of wild-type virus, including several of the late kinetic class. SDS–PAGE and Western blot analysis demonstrated the expression of glycoproteins B and D by 5BlacZ in Vero cells. Initial studies have shown that immunization with 5BlacZ protects guinea pigs from intravaginal HSV-2 challenge. Immunized animals had less severe genital skin disease and reduced replication of the challenge virus in the genital tract during primary infection and reduced episodes of recurrent disease. Thus, HSV-2 ICP8 shows gene regulatory properties similar to those of HSV-1 ICP8, and this HSV-2 ICP8 mutant virus shows a phenotype similar to those of HSV-1 ICP8 mutant strains. Replication-defective mutants of HSV-2 offer a potential vaccine approach for immune intervention against HSV-2 genital disease and latent infection.
Crystals of a 60-amino-acid C-terminal deletion mutant of the herpes simplex virus 1 single-stranded DNA binding protein, ICP8, have been grown by hanging drop vapor diffusion. The colorless crystals grow as thin plates to a maximum size of approximately 0.3 mm x 0.3 mm x 0.05 mm. The space group is P2(1)2(1)2(1) with unit cell constants a = 101.2 A, b = 145.8 A, and c = 162.9 A. There are one or two molecules of ICP8 per asymmetric unit.
Varicella-zoster virus (VZV) transcription is limited in latently infected human ganglia. Note that much of the transcriptional capacity of the virus genome has not been analyzed in detail; to date, only VZV genes mapping to open reading frames (ORFs) 4, 21, 29, 62, and 63 have been detected. ORF 62 encodes the major immediate-early virus transcription transactivator IE62, ORF 29 encodes the major virus DNA binding protein, and ORF 21 encodes a protein associated with the developing virus nucleocapsid. We analyzed the cellular location of proteins encoded by ORF 21 (21p) and ORF 29 (29p), their phosphorylation state during productive infection, and their ability form a protein-protein complex. The locations of both 21p and 29p within infected cells mimic those of their herpes simplex virus type 1 (HSV-1) homologues (UL37 and ICP8); however, unlike these homologues, 21p is not phosphorylated and neither 21p nor 29p exhibits a protein-protein interaction. Transient transfection assays to determine the effect of 21p and 29p on transcription from VZV gene 20, 21, 28, and 29 promoters revealed no significant activation of transcription by 21p or 29p from any of the VZV gene promoters tested, and 21p did not significantly modulate the ability of IE62 to activate gene transcription. A modest increase in IE62-induced activation of gene 28 and 29 promoters was seen in the presence of 29p; however, IE62-induced activation of gene 28 and 29 promoters was reduced in the presence of 21p. A Saccharomyces cerevisiae two-hybrid analysis of 21p indicated that the protein can activate transcription when tethered within a responsive promoter. Together, the data reveal that while VZV gene 21 and HSV-1 UL37 share homology at the nucleic acid level, these proteins differ functionally.
Varicella zoster virus (VZV) is a neurotropic human herpesvirus that infects nearly all humans and causes chickenpox (varicella). After chickenpox, VZV becomes latent in cranial nerve, dorsal root, and autonomic nervous system ganglia along the entire neuraxis. Virus reactivation produces shingles (zoster), characterized by pain and rash usually restricted to 1-3 dermatomes. Zoster is often complicated by postherpetic neuralgia (PHN), pain that persists for months to years after rash resolves. Virus may also spread to the spinal cord and blood vessels of the brain, producing a unifocal or multifocal vasculopathy, particularly in immunocompromised individuals. The increased incidence of zoster in elderly and immunocompromised individuals appears to be due to a VZV-specific host immunodeficiency. PHN may reflect a chronic VZV ganglionitis, and VZV vasculopathy is due to productive virus infection in cerebral arteries. Strategies that might boost host cell-mediated immunity to VZV are discussed, as well as the physical state of viral nucleic acid during latency and the possible mechanisms by which herpesvirus latency is maintained and virus is reactivated. A current summary of varicella latency and pathogenesis produced by simian varicella virus (SVV), the counterpart of human VZV, points to the usefulness of a primate model of natural infection to study varicella latency, as well as the experimental model of intratracheal inoculation to study the effectiveness of antiviral agents in driving persistent varicella virus into a latent state.
All organisms including animal viruses use specific proteins to bind single-stranded DNA rapidly in a non-sequence-specific,
flexible, and cooperative manner during the DNA replication process. The crystal structure of a 60-residue C-terminal deletion
construct of ICP8, the major single-stranded DNA-binding protein from herpes simplex virus-1, was determined at 3.0 Å resolution.
The structure reveals a novel fold, consisting of a large N-terminal domain (residues 9-1038) and a small C-terminal domain
(residues 1049-1129). On the basis of the structure and the nearest neighbor interactions in the crystal, we have presented
a model describing the site of single-stranded DNA binding and explaining the basis for cooperative binding. This model agrees
with the beaded morphology observed in electron micrographs.
We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.
Infected-cell protein 27 (ICP27) is a herpes simplex virus type 1 alpha, or immediate-early, protein involved in the regulation of viral gene expression. To better understand the function(s) of ICP27 in infected cells, we have isolated and characterized viral recombinants containing defined alterations in the ICP27 gene. The mutant virus d27-1 contains a 1.6-kilobase deletion which removes the ICP27 gene promoter and most of the coding sequences, while n59R, n263R, n406R, and n504R are mutants containing nonsense mutations which encode ICP27 molecules truncated at their carboxyl termini. All five mutants were defective for lytic replication in Vero cells. Analysis of the mutant phenotypes suggests that ICP27 has the following regulatory effects during the viral infection: (i) stimulation of expression of gamma-1 genes, (ii) induction of expression of gamma-2 genes, (iii) down regulation of expression of alpha and beta genes late in infection, and (iv) stimulation of viral DNA replication. Cells infected with the mutant n504R expressed wild-type levels of gamma-1 proteins but appeared to be unable to efficiently express gamma-2 mRNAs or proteins. This result suggests that ICP27 mediates two distinct transactivation functions, one which stimulates gamma-1 gene expression and a second one required for gamma-2 gene induction. Analysis of the mutant n406R suggested that a truncated ICP27 polypeptide can interfere with the expression of many viral beta genes. Our results demonstrate that ICP27 has a variety of positive and negative effects on the expression of viral genes during infection.
We have determined the DNA sequence of the long unique region (UL) in the genome of herpes simplex virus type 1 (HSV-1) strain 17. The UL sequence contained 107,943 residues and had a base composition of 66.9% G + C. Together with our previous work, this completes the sequence of HSV-1 DNA, giving a total genome length of 152,260 residues of base composition 68.3% G + C. Genes in the UL region were located by the use of published mapping analyses, transcript structures and sequence data, and by examination of DNA sequence characteristics. Fifty-six genes were identified, accounting for most of the sequence. Some 28 of these are at present of unknown function. The gene layout for UL was found to be very similar to that for the corresponding part of the genome of varicella-zoster virus, the only other completely sequenced alphaherpesvirus, and the amino acid sequences of equivalent proteins showed a range of similarities. In the whole genome of HSV-1 we now recognize 72 genes which encode 70 distinct proteins.
The expression of herpes simplex virus gamma 2 (late) genes is inhibited before the onset of viral DNA replication. We report that the block in the expression of certain gamma 2 genes is relieved, at least in part, by defects in the beta ICP8 protein. We have examined the expression of the gamma 2 gene encoding glycoprotein C (gC) in cells infected with a temperature-sensitive ICP8 mutant. Under conditions in which viral DNA replication is inhibited, cells infected with the ICP8 mutant overproduce the gC family of mRNAs relative to the level observed in cells infected with a wild-type virus. The gC mRNA synthesized in cells infected with the ICP8 mutant virus is correctly initiated and spliced and is translated with the same relative efficiency as in cells infected with a replicating wild-type virus. These results suggest that ICP8 is involved in the negative regulation of gamma 2 genes expressed from parental viral genomes. The level of gC expression was greatest in cells infected with a replicating wild-type virus. These data suggest that DNA replication and genome amplification are not absolute requirements for gamma 2 gene expression but may facilitate full-level expression of these genes.
In the long unique region of the genome of herpes simplex virus type 1 (HSV-1), the genes for DNA polymerase and the major
DNA binding protein are arranged in a head to head manner, with an origin of DNA replication (termed OriL) located between them. This paper reports an 8400 base pair DNA sequence containing both genes and the origin, obtained mostly
by M13/dideoxy analysis of plasmid cloned fragments. Amino acid sequences of the two proteins were deduced. Homologues of
both genes were detected in the genome sequence of the distantly related Epstein-Barr virus (EBV). Arrangement of these HSV-1
and EBV genes differs in genome location and in relative orientation. A part of HSV-1 DNA polymerase was found to be similar
to a sequence in adenovirus 2 DNA polymerase, but the significance of this is unclear. Since a DNA sequence in the locality
of OriL deletes on plasmid cloning, this region was analysed using virus DNA. A palindrome with 72-residue arms was found, which
shows great similarity to the better characterized origin, OriS.
We examined the kinetics and the nature of the association of two herpes simplex virus proteins, the major DNA-binding protein (ICP8) and the major capsid protein (ICP5), with the nuclei of infected cells. We defined a series of stages in the association of the ICP8 protein with the cell nucleus. (i) Immediately after synthesis, the protein was found in the cytoplasmic fraction but associated rapidly with the crude nuclear fraction. (ii) The initial association of ICP8 with the crude nuclear fraction was detergent sensitive but DNase resistant, and, thus, the protein was either bound to structures attached to the outside of the nucleus and had not penetrated the nuclear envelope or was loosely bound in the nucleus, (iii) At intermediate times, a low level of an intermediate form was observed in which the association of ICP8 with the nuclear fraction was resistant to both detergent and DNase treatment. The protein may be bound to the nuclear matrix at this stage. Inhibition of viral DNA synthesis caused the DNA-binding protein to accumulate in this form. (iv) At late times during the chase period, the association of ICP8 with the cell nucleus was resistant to detergent treatment but sensitive to DNase treatment. our results argue that at this stage ICP8 was bound to viral DNA. Thus, nuclear association of the DNA-binding protein did not require viral DNA replication. More important is the observation that there is a series of stages in the nuclear association of this protein, and, thus, there may be a succession of binding sites for this protein in the cell during its movement to its final site of action in the nucleus. The major capsid protein showed some similar stages of association with the cell nucleus but the initial association with the nucleus followed a lag period. Its early association with the crude nuclear fraction was also detergent sensitive but was resistant to detergent treatment at later times. Its association with the cell nucleus was almost completely resistant to DNase treatment at all times. Inhibition of viral DNA replication blocked the nuclear transport of this protein. Thus, these two viral proteins share some stages in nuclear transport, although their requirements for nuclear association are different.
We have resolved two electrophoretic species of the major DNA-binding protein, infected cell polypeptide 8 (ICP8), encoded by herpes simplex virus 1. In pulse-chase experiments, we observed the conversion of the ICP8a form, the slower migrating species, to the faster migrating form, ICP8b. Thus, the two species appear to be related as precursor-product. The conversion was not due to proteolytic cleavage, because higher concentrations of reducing agents in the sample buffer shifted the faster moving form to the slower moving species. Also, the two forms have identical peptide patterns as analyzed by partial proteolysis in sodium dodecyl sulfate. Thus, the faster moving species appears to be a conformational isomer containing intramolecular disulfide bonds. The functional significance of the two forms of the protein is discussed.
Two herpes simplex virus proteins, the major capsid protein and the major DNA binding protein, are specifically localized to the nucleus of infected cells. We have found that the major proportion of these proteins is associated with the detergent-insoluble matrix or cytoskeletal framework of the infected cell from the time of their synthesis until they have matured to their final binding site in the cell nucleus. These results suggest that these two proteins may interact with or bind to the cellular cytoskeleton during or soon after their synthesis and throughout transport into the cell nucleus. In addition, the DNA binding protein remains associated with the nuclear skeleton at times when it is bound to viral DNA. Thus, viral DNA may also be attached to the nuclear framework. We have demonstrated that the DNA binding protein and the capsid protein exchange from the cytoplasmic framework to the nuclear framework, suggesting the direct movement of the proteins from one structure to the other. Inhibition of viral DNA replication enhanced the binding of the DNA binding protein to the cytoskeleton and increased the rate of exchange from the cytoplasmic framework to the nuclear framework, suggesting a functional relationship between these events. Inhibition of viral DNA replication resulted in decreased synthesis and transport of the capsid protein. We have been unable to detect any artificial binding of these proteins to the cytoskeleton when solubilized viral proteins were mixed with a cytoskeletal fraction or a cell monolayer. This suggested that the attachment of these proteins to the cytoskeleton represents the actual state of these proteins within the cell.
The major DNA-binding protein encoded by several temperature-sensitive mutants of herpes simplex virus type 1 was thermolabile for binding to intracellular viral DNA. The ability of DNase I to release this protein from isolated nuclei was used as a measure of the amount of protein bound to viral DNA. This assay was based upon our previous observation that the fraction of herpesviral DNA-binding protein which can be eluted from nuclei with DNase I represents proteins associated with progeny viral DNA (D. M. Knipe and A. E. Spang, J. Virol. 43:314-324, 1982). In this study, we found that several temperature-sensitive mutants encoded proteins which rapidly chased from a DNase I-sensitive to a DNase I-resistant nuclear form upon shift to the nonpermissive temperature. We interpret this change in DNase I sensitivity to represent the denaturation of the DNA-binding site at the nonpermissive temperature and the association with the nuclear framework via a second site on the protein. The DNA-binding activity measured by the DNase I sensitivity assay represents an important function of the protein in viral replication because three of five mutants tested were thermolabile for this activity. A fourth mutant encoded a protein which did not associate with the nucleus at the nonpermissive temperature and therefore would not be available for DNA binding in the nucleus. We also present supportive evidence for the binding of the wild-type protein to intracellular viral DNA by showing that a monoclonal antibody coprecipitated virus-specific DNA sequences with the major DNA-binding protein.
We have examined the effect of temperature-sensitive mutations in the herpes simplex virus 1 DNA-binding protein gene on viral gene expression. We have found that at the nonpermissive temperature, the synthesis of certain immediate early, early, and late viral polypeptides was greater in cells infected with the temperature-sensitive mutants than in cells infected with the wild-type virus. This effect was independent of the requirement for this viral protein for viral DNA replication. The altered rate of synthesis of viral proteins was due to a thermolabile gene product. Cells infected with these mutants at the permissive temperature and then shifted to the nonpermissive temperature exhibited enhanced levels of viral gene expression. The addition of actinomycin D at the time of the temperature shift prevented the alteration in viral protein synthesis. Therefore, continuing transcription is required for this change in gene expression. Northern blot analysis of cytoplasmic RNA showed that the steady-state level of specific viral transcripts expressed from parental virus genomes was greater in cells infected by these mutants at the nonpermissive temperature. These results indicate that the major DNA-binding protein of herpes simplex virus type 1 acts as a negative regulator of viral gene expression by affecting the abundance of cytoplasmic viral mRNAs.
The structural genes encoding the herpes simplex virus type 1 glycoprotein B and the major DNA-binding protein ICP8 have been mapped previously within the EcoRI-F restriction fragment (map coordinates 0.314 to 0.420) of the viral genome. In this study the mRNAs transcribed from these DNA sequences were identified by hybridization selection of 32P-labeled RNA and by Northern blot analysis of polyadenylated cytoplasmic RNA. A 3.4-kilobase RNA was the major mRNA homologous to the DNA sequences between coordinates 0.343 and 0.386 in which mutations in the glycoprotein B gene have been mapped. A 4.5-kilobase RNA was the major mRNA homologous to the viral DNA sequences between coordinates 0.361 and 0.417 in which mutations in the ICP8 gene have been mapped. Hybridization-selected mRNAs were translated in vitro to determine the primary translation products encoded in each region. The glycoprotein B- and ICP8-specific polypeptides were identified by immunoprecipitation with specific antisera. The translation products encoded by the glycoprotein B gene were 103,000 and 99,000 in molecular weight. The translation products encoded by the ICP8 gene were 125,000 and 122,000 in molecular weight.
During herpes simplex virus infection, expression of the viral DNA polymerase (pol) gene is regulated temporally as an early (beta) gene and is additionally down-regulated at late times at the level of translation (D. R. Yager, A. I. Marcy, and D. M. Coen, J. Virol. 64:2217-2225, 1990). To examine the role of viral DNA synthesis in pol regulation, we studied pol expression during infections in which viral DNA synthesis was blocked, either by using drugs that inhibit Pol or ribonucleotide reductase or by using viral mutants with lesions in either the pol or a primase-helicase subunit gene. Under any of these conditions, the level of cytoplasmic pol mRNA was reduced. This reduction was first seen at approximately the time DNA synthesis begins and, when normalized to levels of other early mRNAs, became as great as 20-fold late in infection. The reduction was also observed in the absence of the adjacent origin of replication, oriL. Thus, although pol mRNA accumulated as expected for an early gene in terms of temporal regulation, it behaved more like that of a late (gamma) gene in its response to DNA synthesis inhibition. Surprisingly, despite the marked decrease in pol mRNA in the absence of DNA synthesis, the accumulation of Pol polypeptide was unaffected. This was accompanied by loss of the normal down-regulation of translation of pol mRNA at late times. We suggest a model to explain these findings.
Herpes simplex virus replicates its DNA within nuclear structures called replication compartments. In contrast, in cells in which viral DNA replication is inhibited, viral replication proteins localize to punctate structures called prereplicative sites. We have utilized viruses individually mutated in each of the seven essential replication genes to assess the function of each replication protein in the assembly of these proteins into prereplicative sites. We observed that four replication proteins, UL5, UL8 UL52, and UL9, are necessary for the localization of ICP8 (UL29) to prereplicative sites natural infection conditions. Likewise, four of the seven viral DNA replication proteins, UL5, UL52, UL9, and ICP8, are necessary for the localization of the viral DNA polymerase to prereplicative sites. On the basis of these results, we present a model for prereplicative site formation in infected cells in which the helicase-primase components (UL5, UL8, and UL52), the origin-binding protein (UL9), and the viral single-stranded DNA-binding protein (ICP8) assemble together to initiate the process. This is followed by the recruitment of the viral polymerase into the structures, a step facilitated by the polymerase accessory protein, UL42. Host cell factors can apparently substitute for some of these viral proteins under certain conditions, because the viral protein requirements for prereplicative site formation are reduced in transfected cells and in infected cells treated with drugs that inhibit DNA synthesis.
The major DNA-binding protein or infected cell protein 8 (ICP8) encoded by herpes simplex virus exhibits multiple interactions with the cell nucleus in that it interacts with the host cell nuclear matrix and viral DNA molecules as sequential stages in its maturational process (M. P. Quinlan, L. B. Chen, and D. M. Knipe (1984), Cell 36, 857–868). To define the portion(s) of ICP8 required for DNA binding, we have fine-mapped and identified the sequence changes in mutant genes causing changes in the protein that affect DNA binding. These mutations lead to amino acid changes between residues 348 and 450 of ICP8. Construction of a mutant ICP8 gene specifically altered at residues 499 and 502 led to a gene product that was also defective in a nuclear function. Thus, at least part of the region of ICP8 from residues 348 to 4.50 is required for DNA binding by ICP8. This portion of the protein may be involved in binding to DNA or forming intermolecular contacts needed for cooperative DNA binding. If this region is directly involved in binding of the protein to DNA, the most likely structure predicted for this region involves folding of β-strands to form a channel for binding to a nucleotide chain.
Phosphonoacetate is a highly specific inhibitor of herpes simplex virus-induced DNA polymerase. Sensitivity of herpesvirus type 1 or type 2 induced DNA polymerase to the drug was similar. However, DNA polymerases from other sources such as the host cells (Wi-38), Micrococcus luteus, and hepatitis B virus were highly resistant. In addition, Escherichia coli RNA polymerase and reverse transcriptase of Rous sarcoma virus were also insensitive to the drug. Enzyme kinetic studies showed that inhibition was noncompetitive with respect to deoxyribonucleotide triphosphates. The Ki value was about 0.45 muM. The apparent Km values for dTTP, dATP, dCTP, and dGTP were 0.71, 0.75, 0.42, and 0.39 muM, respectively. The base composition of template has no profound effect on the extent of inhibition. The drug caused uncompetititve inhibition with respect to template which indicated that phosphonoacetate did not bind directly to template DNA. Results are presented which suggest that phosphonoacetate did not affect the formation of the enzyme-DNA complex but probably inhibited the elongation step of DNA polymerase reaction.
In cells infected with herpes simplex virus, HSV-I, newly synthesized polypeptides accumulated in the nucleus at different rates, which did not change during the first 6 h after infection. Canavanine, an arginine analogue, prevented the nuclear accumulation of ICP (infected cell polypeptides) 5 and 8 and azetidine, a proline analogue, prevented that of ICP 5 and 7. The transfer of polypeptides to the nucleus was inhibited at 4 degrees C but not by dinitrophenol. Some of the nuclear polypeptides could be released by washing isolated nuclei with hypertonic salt solutions. ICP 17 was particularly sensitive to high salt treatment while ICP 5 and II were resistent. ICP 4b, a modified form of the alpha polypeptide ICP 4, was released by EDTA, and the detergent NP40 removed ICP II. Treatment of nuclei with DNase selectively reduced the amount of bound alpha polypeptides ICP 4c (the second modified form of ICP 4), 0 and 27 as well as ICP 8 and 25. Nuclei isolated from infected or uninfected cells and incubated in labelled cytoplasmic extracts took up primarily ICP 8 and 32. Alpha polypeptides were taken up to a lesser extent and ICP 6 and 10 were excluded. It is concluded that affinities for various constituents of host cell nuclei are likely to determine the nuclear accumulation of specific virus polypeptides.
This paper deals with control of mRNA levels, assayed by in vitro translation, in cells infected with herpes simplex virus type 1 (HSV-1). A particularly useful marker has been pyrimidine deoxyribonucleoside kinase (dPyK) mRNA, for which the enzymatically active product can be assayed quantitatively. Cells infected with the HSV-1 temperature-sensitive mutant tsK at the nonpermissive temperature (38.5 degrees C) or with wild-type HSV-1 in the continuous presence of cycloheximide contained no detectable dPyK mRNA. Upon temperature shift-down of tsK-infected cells to 31 degrees C, dPyK mRNA was produced, and this event was inhibited by actinomycin D but not cycloheximide. This result demonstrated that the defective polypeptide in tsK-infected cells was involved in transcription of the dPyK gene and could regain activity at 31 degrees C. Because tsK-infected cells synthesized mainly immediate early polypeptides at 38.5 degrees C, the involvement of this polypeptide class in synthesis of dPyK mRNA was investigated. Analysis of the kinetics of inductions of dPyK mRNA indicated that the temperature-sensitive lesion in tsK lies in an immediate early polypeptide which is directly responsible for activation of the dPyK gene at the transcriptional level.
Previous studies have shown that the herpes simplex virus type 1 (HSV-1) mutant tsK has a temperature-sensitive lesion in an immediate early polypeptide whose function is to induce synthesis of new viral transcripts, including mRNA, for pyrimidine deoxyribonucleoside kinase. The studies presented here examine the properties of immediate early polypeptides in wild-type HSV-1 and tsK-infected cells at 31 and 38.5°C. The overall pattern of immediate early protein synthesis was similar in wild-type HSV-1 and tsK-infected cells when radiolabeled with [35S]methionine or 14C-amino acid mixture. Further investigation, however, revealed two aberrant properties of the polypeptide V(mw) 75 in tsK-infected cells at 38.5°C. Upon cell fractionation, large amounts of this polypeptide were recovered in the cytoplasmic fraction, in contrast to tsK-infected cells at 31°C or wild-type HSV-1 infected cells at either temperature. Furthermore, at 38.5°C tsK-induced V(mw) 175 was not processed normally to forms of lower electrophoretic mobility. Both of these defects were reversible upon downshift of tsK-infected cells, even in the absence of further protein synthesis, but were not observed in cells infected with a revertant of tsK. Coinfection of tsK-infected cells with wild-type HSV-1 did not alleviate these lesions, suggesting that they resulted from an abnormal V(mw) 175 polypeptide rather than from a defective processing enzyme. Temperature upshift of tsK-infected cells caused reversion of V(mw) 175 to the mutant form. The progression to synthesis of late polypeptides was also arrested; therefore, a functional lesion was also reversible upon temperature changes between 31 and 38.5°C during the early stages of infection. The identification of a polypeptide with abnormal properties in tsK-infected cells and the demonstration that these properties, and the functional lesion, are reversible may provide an important system for investigation HSV-1 transcriptional control.
The purpose of this investigation was to identify and characterize the regulatory elements involved in the transcriptional activation of the beta gamma (leaky-late or gamma 1) genes of herpes simplex virus type 1 (HSV-1) by using the major capsid protein (VP5 or ICP5) gene as model. Gel mobility shift assays with nuclear extracts from uninfected and infected HeLa cells enabled us to identify two major protein-DNA complexes involving the VP5 promoter. The mobilities of these two complexes remained unaltered, and no unique complexes were observed when infected cell nuclear extracts were used. DNase I and orthophenanthroline-Cu+ footprint analyses revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, we tested the effect of mutations in this region by using transient expression of a cis-linked chloramphenicol acetyltransferase gene. Deletion of the above sequence resulted in a seven- to eightfold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV-1 immediate-early genes. From these results, we conclude that the LBS sequence and a cellular factor(s) are involved in the transactivation of the VP5 gene. A search of published gene sequences revealed that sequences related to the LBS exist in a number of other HSV-1, cytomegalovirus, retrovirus, and cellular promoters. Sequence homologies of binding sites and results of unpublished competition binding studies suggest that this leaky-late binding factor may be related to, or the same as, a ubiquitous cellular transcriptional factor called YY1 or common factor-1 (also known as NF-E1, delta, and UCRBP).
Gene expression by herpes simplex virus type 1 (HSV-1) results in the synthesis of three temporal classes of viral proteins. The three classes of viral proteins are expressed in a cascade manner of sequential dependency. The molecular mechanisms that account for the HSV-1 protein synthesis cascade are poorly understood. In order to provide a detailed description of the metabolic levels at which HSV-1 protein synthesis is regulated, we have measured transcription rates and mRNA accumulation levels for 11 HSV-1 genes. These measurements were made over a time-course of infection in the presence or absence of metabolic inhibitors of either viral protein synthesis or viral DNA synthesis. Our observations show that the protein synthesis cascade of HSV-1 is established as a consequence of mechanisms that regulate both the transcription and accumulation of viral messenger RNA.
Eukaryotic DNA synthesis is thought to occur in multienzyme complexes present at numerous discrete sites throughout the nucleus. We demonstrate here that cellular DNA replication sites identified by bromodeoxyuridine labeling are relocated in cells infected with herpes simplex virus such that they correspond to viral prereplicative structures containing the HSV DNA replication protein, ICP8. Thus components of the cellular DNA replication apparatus are present at viral prereplicative sites. Mutant virus strains expressing defective ICP8 do not alter the pattern of host cell DNA replication sites, indicating that functional ICP8 is required for the redistribution of cellular DNA replication complexes. This demonstrates that a specific protein molecule can play a role in the organization of DNA replication proteins at discrete sites within the cell nucleus.
The wild-type herpes simplex virus 1 genome consists of two components, L and S, which invert relative to each other, giving rise to four isomers. Previously we reported the construction of a herpes simplex virus 1 genome, HSV-1(F)I358, from which 15 kilobase pairs of DNA spanning the junction between L and S components were deleted and which no longer inverted (Poffenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2690-2694, 1983). Further studies on the structure of HSV-1(F)I358 revealed the presence of two submolar populations among packaged DNA. The first, comprising no more than 10% of total packaged DNA, consisted of defective genomes with a subunit size of 36 kilobase pairs. The results suggest that this population arose by recombination through a directly repeated sequence inserted in place of the deleted L-S junction. The second minor population consisted of HSV-1(F)I358 DNA linked head-to-tail. Analyses of the structure of HSV-1(F)I358 DNA after infection indicated that the fraction of total DNA linked head-to-tail increased to approximately 40 to 50% within 30 min after exposure of cells to virus. The formation of head-to-tail linkages did not require de novo protein synthesis. Our interpretation of the results is that the termini of full-length DNA molecules are held together during packaging, that a small fraction of the termini is covalently linked during or after packaging, and that the remainder is covalently joined after the release of viral DNA from the infecting virus by either host or viral factors introduced into the cell during infection.
We have determined the complete DNA sequence of the short unique region in the genome of herpes simplex virus type 1, strain 17, and have interpreted it in terms of messenger RNAs and encoded proteins. The sequence contains variable regions whose length differs between DNA clones. The clones used for most of the analysis gave a short unique length of 12,979 base-pairs. We consider that this region contains 12 genes, which are expressed by mRNAs which have separate promoters, but may share 3'-termination sites, so that all but two mRNAs belong to one of four 3'-coterminal "families": 79% of the sequence is considered to be polypeptide coding. One pair of genes has an extensive out-of-frame overlap of coding sequences. The proteins encoded in the short unique region include two immediate-early species, two virion surface glycoproteins, and a DNA-binding species. Six of the genes have little or no previous characterization. From the nature of the amino acid sequences predicted for their encoded proteins, we deduce that several of these proteins may be membrane-associated.
We have used an in vitro nuclear run-off assay to measure the levels of transcription of specific herpes simplex virus genes at different times during a lytic infection. We analyzed the effects of inhibition of DNA replication and of defects in two herpes simplex virus regulatory proteins on the transcription of these genes. We present evidence that the transcription of the alpha ICP4 gene is negatively regulated during a lytic infection. The regulation of ICP4 gene transcription requires the beta protein ICP8 (where ICP = infected cell polypeptide). Transcription of the beta ICP8, gamma 1 ICP5, and gamma 2 glycoprotein C (gC) genes was dependent on ICP4, and transcription of the gamma 2gC gene was strongly inhibited when DNA replication was blocked. Defects in ICP8 also resulted in increased levels of transcription of the ICP4, ICP8, ICP5, and gC genes from parental viral genomes. Our results suggest that ICP8 may be important in maintaining the highly ordered cascade of viral gene expression.
Several laboratories have shown that transfected plasmid DNAs containing either of the two known origins of herpes simplex virus (HSV) DNA replication, oriS or oriL, are replicated in HSV-1-infected cells or in cells cotransfected with virion DNA. I have found that HSV-1 (KOS) DNA digested to completion with the restriction enzyme Xba I is as efficient as intact viral DNA in supporting the in vivo replication of cotransfected plasmids containing oriS. On the basis of this result, several of the Xba I restriction fragments of HSV-1 DNA were cloned into the plasmid vector pUC19, and combinations of cloned DNAs were tested for their ability to supply the trans-acting functions required for HSV origin-dependent replication. A combination of five cloned fragments of HSV-1 can supply all of the necessary functions: Xba I C (coordinates 0.074-0.294), Xba I F (coordinates 0.294-0.453), Xba I E (coordinates 0.453-0.641), Xba I D (coordinates 0.641-0.830), and EcoRI JK (coordinates 0.0-0.086; 0.830-0.865). Transient plasmid replication in this system is dependent on the presence of either oriS or oriL in cis. The plasmid containing Xba I F can be replaced by two smaller plasmids, one of which contains only the gene for the HSV-encoded DNA polymerase, and the other of which contains only the gene for the major DNA binding protein (ICP8). Thus, plasmid DNA replication in this system depends on two of the genes known from genetic studies to be essential for viral DNA replication in infected cells. This system defines a simple complementation assay for cloned fragments of HSV DNA that contain other genes involved in viral DNA replication and should lead to the rapid identification of all such genes.
To minimize the contribution of residual activity associated with the temperature-sensitive (ts) form of ICP8 specified by available ts mutants, deletion mutations in this gene were constructed. Cells permissive for the generation and propagation of ICP8 deletion mutants were first obtained. Vero cells were cotransfected with pKEF-P4, which contains the gene for ICP8, and pSV2neo or a hybrid plasmid containing the G418 resistance gene linked to pKEF-P4. Of the 48 G418-resistant cell lines, 21 complemented ICP8 ts mutants in plaque assays at the nonpermissive temperature. Four of these were examined by Southern blot analysis and shown to contain 1 to 3 copies of the ICP8 gene per haploid genome equivalent. Cell line U-47 was used as the permissive host for construction of ICP8 deletion mutants. In addition to cell lines which complemented ts mutants, two lines, U-27 and U-35, significantly inhibited plaque formation by wild-type virus, contained 30 and 100 copies of the ICP8 gene per haploid genome equivalent, respectively, and expressed large amounts of ICP8 after infection with wild-type virus. At low but not high multiplicities of infection, this inhibition was accompanied by underproduction of viral polypeptides of the early, delayed-early, and late kinetic classes. For construction of deletion mutants, a 780-base-pair XhoI fragment was deleted from pSG18-SalIA, a plasmid which contains the gene for ICP8, to yield pDX. U-47 cells were then cotransfected with pDX and infectious wild-type DNA. Mutant d61, isolated from the progeny of cotransfection, was found to contain both the engineered deletion in the ICP8 gene and an oriL-associated deletion of approximately 55 base pairs. Because d61 contained two mutations, a second mutant, d21, which carried the engineered ICP8 deletion but an intact oriL, was constructed by cotransfection of U-47 cells with wild-type DNA and an SalI-KpnI fragment purified from pDX. Phenotypic analysis of d21 and d61 revealed that they were similar in all properties examined: both exhibited efficient growth in U-47 cells but not in Vero cells; both induced the synthesis of an ICP8 polypeptide which was smaller than the wild-type form of the protein and which, unlike the wild-type protein, was found in the cytoplasm and not the nucleus of infected Vero cells; and nonpermissive Vero cells infected with either mutant failed to express late viral polypeptides.
The rate of synthesis in vivo and the steady-state level of mRNA of four "model" herpes simplex virus type 1 (HSV-1) genes were measured as a function of high levels of alpha-gene products. The genes studied were ICP4 (alpha), deoxy-UTPase (beta), VP5 (beta gamma), and glycoprotein C (gC, gamma). Accumulation of high levels of alpha proteins was accomplished either by infection with an HSV-1 mutant, temperature-sensitive in ICP4 (ts606) at the nonpermissive temperature then shift-down to permissive temperature, or by infection with wild-type virus under cycloheximide blockage of protein synthesis followed by release. Compared to RNA expression in normal infections, beta gamma and gamma transcription rates were both transiently stimulated under the experimental conditions employed. The greatest effect was seen with the gamma-gC mRNA transcription rates. In addition, at nonpermissive temperatures with ts 606, the amount of expression of gC mRNA was significantly increased over normal early levels, in contrast to the case with the VP5 transcript. The impact of such results on models of HSV gene expression in vivo are discussed.
This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The positions on the DNA
sequence corresponding to the 5′ and 3′ termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly
more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of
the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk
gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the
5′ terminus of tk mRNA to a USA stop codon ∼70 nucleotides from the poly-A addition site. The results of these experiments
indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent
to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.
The herpes simplex virus type 1 (HSV-1) major capsid protein VP5 gene (UL19) is expressed with beta gamma (gamma 1 [leaky late]) kinetics. We have previously described the construction of recombinant HSV-1 in which the VP5 promoter was engineered to control the expression of the bacterial beta-galactosidase gene as a reporter (C.-J. Huang, S. A. Goodart, M. K. Rice, J. F. Guzowski, and E. K. Wagner, J. Virol. 67:5109-5116, 1993). Here we describe further mutational analysis in recombinant viruses. We have precisely defined the boundaries of the VP5 promoter and identified two regions important for both the level and the kinetics of expression. The 5' boundary was located at -48 relative to the initiation site of transcription by analyzing a series of nested deletions in the upstream sequence, and although a number of cis-acting sites influencing transient expression have been identified upstream of this point, these sites have no role in promoter activity during productive infection. Deletion of an Sp1-binding site located between -48 and the TATA box at -30 greatly reduced VP5 promoter activity late but not early after infection. A cis-acting element whose sequence resembles the human immunodeficiency virus type 1 initiator was located between -2 and +10 in the VP5 sequence by characterizing a series of deletions and site-directed block mutations downstream the TATA box. This element defines the 3' limit of the VP5 promoter, and like the upstream element, disruption of this element also inhibited promoter activity late in the productive cycle.
Correct intranuclear localization of herpes Characterization simplex virus DNA polymerase requires the viral ICP8 DNA-binding of two conformational forms of the major DNA-binding protein en-protein
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