[Show abstract][Hide abstract] ABSTRACT: Herpes simplex virus type 1 (HSV-1)-based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4-). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4- vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the nonreplicating vector largely resembles mock infection.
Journal of NeuroVirology 09/2009; 15(5-6):411-24. DOI:10.3109/13550280903473452 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Herpes simplex virus type 1 (HSV-1) uses multicomponent mechanisms for binding, penetration, and cell-to-cell passage. These processes are affected by polysulfonated compounds. In this paper we have addressed the question of whether the same or different interactions of HSV-1 with polysulfonated compounds are involved in binding, penetration, and passage. For this, we have compared the inhibitory dose-response for a series of polysulfonated and cationic compounds known to block HSV-1 infections. These comparisons were done at the level of binding, penetration, and cell-to-cell passage. Variations in the parameters of the dose-response curves - IC(50) and Hill coefficients (n (H)) - are consistent with HSV-1 having multiple interactions with sulfonated cellular components in all these processes. Some of the interactions seem to be common to the three processes, while others are particular for each one.
[Show abstract][Hide abstract] ABSTRACT: The genomes of human herpes virus type-1 and type-2 share a high degree of sequence identity; yet, they exhibit important differences in pathology in their natural human host as well as in animal host and cell cultures. Here, we report the comparative analysis of the time and relative abundance profiles of the transcription of each virus type (their transcriptomes) using parallel infections and microarray analysis using HSV-1 probes which hybridize with high efficiency to orthologous HSV-2 transcripts. We have confirmed that orthologous transcripts belong to the same kinetic class; however, the temporal pattern of accumulation of 4 transcripts (U(L)4, U(L)29, U(L)30, and U(L)31) differs in infections between the two virus types. Interestingly, the protein products of these transcripts are all involved in nuclear organization and viral DNA localization. We discuss the relevance of these findings and whether they may have potential roles in the pathological differences of HSV-1 and HSV-2.
[Show abstract][Hide abstract] ABSTRACT: The design and construction of a long (75-mer) oligonucleotide-based DNA microarray for herpes simplex virus type 2 transcripts is described. This array is utilized to generate an analysis of HSV-2 transcript abundance as a function of conditions of infection of human cells, and global patterns of HSV-2 transcript abundance are compared with those for HSV-1. General similarities in patterns along with notable differences in specific details are noted. These results reveal a marked conservation in the program of gene activity between phenotypically diverged strains.
Methods in Molecular Biology 02/2005; 292:423-48. · 1.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent advances in DNA and protein microarray methodology and the emerging technology of cell-based sensors have massively increased the speed and sensitivity with which we can detect viral infections. The advantages of the multi-parameter microarray technologies could be combined with the speed and sensitivity of cell-based systems to give 'cell-omic' sensors.
[Show abstract][Hide abstract] ABSTRACT: To investigate the impact of stress kinase p38 activation on HSV-1 transcription, we performed a global transcript profile analysis of viral mRNA using an oligonucleotide-based DNA microarray. RNA was isolated from Vero cells infected with the KOS strain of HSV-1 in the presence or absence of SB203580, a pyridinyl imidazole inhibitor of p38. Under conditions that eliminated ATF2 activation but had no effect on c-Jun, and reduced virus yield by 85-90%, no effect on accumulation of viral IE, DE, or L transcripts was observed by array analysis or selected Northern blot analysis at 2, 4, and 6 h post infection. Results of array data from cells infected with the ICP27 mutant d27-1 in the presence or absence of SB203580 only reflected the known restricted transcription phenotype of the ICP27 mutant. This result is consistent with a role for p38 activation on virus replication lying downstream of the essential role of ICP27 in DE and perhaps late transcription regulation. No effect of SB203580 on transcription was detected after infection with the ICP0 mutant 7134, at 0.5 or 5.0 PFU/cell, though decreases in the rate of accumulation of all kinetic classes of mRNA could be detected, relative to wt virus. These results indicate that inhibiting p38 activity in Vero cells, while significantly reducing wt virus yield, demonstrated no obvious impact on the program of viral transcription.
[Show abstract][Hide abstract] ABSTRACT: We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.
Journal of Virology 11/2004; 78(19):10470-8. DOI:10.1128/JVI.78.19.10470-10478.2004 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The latency-associated transcript (LAT) is required for efficient reactivation of herpes simplex virus type 1 from latent infection in the rabbit eye model, but LAT's mechanism of action is unknown. In addition to reactivation, the LAT region seems to correspond to multiple functions, with some LAT deletion mutants exhibiting increased virulence, increased neuronal death, and restricted establishment of latency. While a LAT promoter deletion mutant (17DeltaPst) seems to be primarily restricted in reactivation in the rabbit, subtle effects on virulence or the establishment of latency cannot be precluded at the normal high levels of virus inoculum used in the rabbit model. Since such additional LAT phenotypes may be more evident with lower doses of virus, we evaluated the influence of initial viral inoculum and LAT expression on the progression of acute infection and the establishment of latency. We have assayed both virus recovery rates and viral genome loads in rabbit corneas and trigeminal ganglia. Our results show that (i) in the corneas and trigeminal ganglia, the maximum amount of virus present during acute infection is independent of the LAT genotype and inoculum dose, although greater viral yields are obtained earlier with higher inoculum doses, and (ii) the range in numbers of latent genomes detected in the ganglia is independent of the inoculum dose and the LAT genotype and therefore no difference in establishment of latency is observed.
Journal of Virology 06/2004; 78(10):5038-44. DOI:10.1128/JVI.78.10.5038-5044.2004 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During productive infection by herpes simplex virus 1 (HSV-1), viral gene expression occurs in a temporally regulated cascade in which transcription of the viral immediate-early (IE) genes is strongly stimulated by the virion protein VP16. We have employed an oligonucleotide microarray to examine the effect of VP16 mutations on the overall pattern of viral gene expression following infection of HeLa cells. This microarray detects essentially all HSV-1 transcripts with relative and absolute levels correlating well with known kinetics of expression. This analysis revealed that deletion of the VP16 activation domain sharply reduced overall viral gene expression; moreover, the pattern of this reduced expression varied greatly from the pattern of a wild-type (wt) infection. However, when this mutant virus was delivered at a high multiplicity of infection or in the presence of the cellular stress inducer hexamethylene bisacetamide, expression was largely restored to the wt levels and pattern. Infection with virions that deliver wt VP16 protein at the start of infection but synthesize only truncated VP16 resulted in a normal kinetic cascade. This suggests that newly synthesized VP16 does not play a significant role in the expression of later classes of transcripts. The VP16 activation domain comprises two subregions. Deletion of the C-terminal subregion resulted in minimal changes in the level and profile of gene expression compared to a normal (wt) cascade. In contrast, deletion of the N-terminal subregion reduced the overall expression levels and skewed the relative levels of IE transcripts but did not significantly alter the kinetic pattern of early and late transcript expression. We conclude that the general activation of IE gene transcription by VP16, but not the specific ratios of IE transcripts, is necessary for the subsequent ordered expression of viral genes. Moreover, this report establishes the feasibility of microarray analysis for globally assessing viral gene expression programs as a function of the conditions of infection.
Journal of Virology 01/2003; 76(24):12758-74. DOI:10.1128/JVI.76.24.12758-12774.2002 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A relatively crude preparation of herpes simplex virus was rapidly visualized by atomic force microscopy after exposure to conditions that produced gradual degradation of the virions. Images were obtained of 1) the intact, enveloped virus; 2) the underlying capsid with associated tegument proteins along with fragments of the membrane; 3) the capsomeres composing the capsid and their surface arrangement; 4) damaged and partially degraded capsids with missing capsomeres; and 5) the DNA extruded from damaged virions. These images provide a unique perspective on the structures of individual virus particles. Atomic force microscopy can thus be used as a diagnostic tool to provide a rapid way to obtain high-resolution images of human pathogens from crude preparations. It is a useful technique that complements X-ray-based structure determination, cryo-electron microscopy techniques, and optical microscopies in the field of molecular pathogenesis.
American Journal Of Pathology 07/2002; 160(6):1959-66. DOI:10.1016/S0002-9440(10)61145-5 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dimethyl sulfoxide (DMSO) is frequently used at a concentration of up to 95% in the formulation of antiherpetic agents because of its properties as a skin penetration enhancer. Here, we have analyzed the effect of DMSO on several parameters of Herpes Simplex Virus replication.
Productive infection levels of HSV-1 were determined by plaque assay or by reporter gene activity, and its DNA replication was estimated by PCR. Transcript levels were evaluated with HSV-specific DNA micro-arrays.
DMSO blocks productive infection in vitro in different cell types with a 50% inhibitory concentration (IC50) from 0.7 to 2% depending upon the multiplicity of infection. The concentration dependence exhibits a Hill coefficient greater than 1, indicating that DMSO blocks productive infection by acting at multiple different points (mechanisms of action) with positive cooperativity. Consistently, we identified at least three distinct temporal target mechanisms for inhibition of virus growth by DMSO. At late stages of infection, DMSO reduces virion infectivity, and markedly inhibits viral DNA replication. A third mode of action was revealed using an oligonucleotide-based DNA microarray system for HSV. These experiments showed that DMSO reduced the transcript levels of many HSV-1 genes; including several genes coding for proteins involved in forming and assembling the virion. Also, DMSO markedly inhibited some but not all early transcripts indicating a previously unknown mode for inhibiting the early phase of HSV transcription-replication cycle.
These observations suggest that DMSO itself may have a role in the anti-herpetic activity of formulations utilizing it as a dispersant.
[Show abstract][Hide abstract] ABSTRACT: While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a "leaky-late" protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (U(L)38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.
Journal of Virology 04/2002; 76(5):2199-205. DOI:10.1128/jvi.76.5.2199-2205.2002 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have localized the region of the latency-associated transcript (LAT) of HSV-1 responsible for epinephrine-induced reactivation in the rabbit eye model to the first 1.5 kb of the primary transcript. This region extends from the 5prime prime or minute exon of the primary LAT transcript through the 5prime prime or minute half of the LAT 2.0-kb intron. To determine whether the 5prime prime or minute end of the LAT intron contributes to the induced reactivation phenotype, three recombinant viruses containing deletions within this portion of the LAT intron were constructed. The three recombinants, containing deletions spanning a combined region of 969 bp at the 5prime prime or minute end of the LAT intron, reactivated with the wild-type frequency of 17syn+. These results indicate that the elements governing induced reactivation reside within the first 699 bp of the primary LAT transcript encoding the 5prime prime or minute LAT exon.
[Show abstract][Hide abstract] ABSTRACT: Neuronal immediate-early gene (IEG) expression is regulated by synaptic activity and plays an important role in the neuroplastic mechanisms critical to memory consolidation. IEGs can be divided into two functional classes: (1) regulatory transcription factors (RTFs), which can broadly influence cell function depending on the "downstream" genes they regulate, and (2) "effector" proteins, which may directly modulate specific cellular functions. The objective of the current study was to determine whether the expression of an effector IEG (Arc) was similar to, or different from, that of two well characterized RTF IEGs (c-fos and zif268) after learning. IEG RNA levels from rats trained in spatial and nonspatial water tasks were determined using RNase protection assays and in situ hybridization. Overall, the regulation of the three IEGs was similar in the hippocampus and the entorhinal and primary visual cortices. Consequently, IEG RNA levels were positively correlated within a structure. By contrast, Arc and zif268 RNA levels were not correlated or only weakly correlated across structures, although c-fos RNA levels were moderately correlated across structures. Arc RNA expression differed from that of zif268 and c-fos in two regards: (1) hippocampal Arc RNA levels were correlated with learning of the hippocampal-dependent spatial, but not hippocampal-independent cued response, water task, and (2) Arc RNA levels in the hippocampus and entorhinal cortex increased after spatial reversal learning relative to an asymptotic performance group. Thus, although the expression of Arc, zif268, and c-fos exhibited many similarities, Arc was most responsive to differences in behavioral task demands.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 08/2001; 21(14):5089-98. · 6.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A class of strict late Herpes Simplex Virus Type 1 (HSV-1) promoters contains a conserved sequence element (termed the downstream activation sequence, DAS) located downstream of the transcription start site. These DAS-containing promoters also require both a TATA box and an initiator element for maximal levels of transcription. In this communication, we demonstrate that the downstream promoter element (DPE) found on a class of Drosophila TATA-less promoters and known to bind the homologue of human TAF(II)70 (a component of TFIID), can functionally substitute for DAS in the context of the strict late UL38 promoter in spite of no obvious sequence similarity. Although Drosophila DPE-containing promoters do not require a TATA box, the element does not remove the requirement for a TATA box when functioning in the HSV promoter. Next, we demonstrate that hTAF(II)70, interacts in a sequence specific manner with DAS as predicted from the fact that DPE binds Drosophila TBP. These results suggest that multiple TFIID/promoter interactions are important in the activation of HSV-1 late gene expression upon viral DNA replication. We propose that such interactions could be favored upon viral DNA replication since TFIID concentrates to viral transcription foci that form during the later stages of infection.
[Show abstract][Hide abstract] ABSTRACT: More than 100 transcripts of various abundances and kinetic classes are expressed during phases of productive and latent infections by herpes simplex virus (HSV) type 1. To carry out rapid global analysis of variations in such patterns as a function of perturbation of viral regulatory genes and cell differentiation, we have made DNA microchips containing sets of 75-mer oligonucleotides specific for individual viral transcripts. About half of these are unique for single transcripts, while others function for overlapping ones. We have also included probes for 57 human genes known to be involved in some aspect of stress response. The chips efficiently detect all viral transcripts, and analysis of those abundant under various conditions of infection demonstrates excellent correlation with known kinetics of mRNA accumulation. Further, quantitative sensitivity is high. We have further applied global analysis of transcription to an investigation of mRNA populations in cells infected with a mutant virus in which the essential immediate-early alpha27 (U(L)54) gene has been functionally deleted. Transcripts expressed at 6 h following infection with this mutant can be classified into three groups: those whose abundance is augmented (mainly immediate-early transcripts) or unaltered, those whose abundance is somewhat reduced, and those where there is a significant reduction in transcript levels. These do not conform to any particular kinetic class. Interestingly, levels of many cellular transcripts surveyed are increased. The high proportion of such transcripts suggests that the alpha27 gene plays a major role in the early decline in cellular gene expression so characteristic of HSV infection.
Journal of Virology 12/2000; 74(21):9916-27. DOI:10.1128/JVI.74.21.9916-9927.2000 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The HSV-1 VP5 and VP16 transcripts are expressed with leaky-late (gamma1) kinetics and reach maximal levels after viral DNA replication. While the minimal VP5 promoter includes only an Sp1 site at -48, a TATA box at -30, and an initiator (Inr) element at the cap site, here we show that elements upstream of -48 can functionally compensate for the mutational loss of the critical Sp1 site at -48. To determine whether this is a general feature of leaky-late promoters, we have carried out a detailed analysis of the VP16 promoter in the context of the viral genome at the gC locus. Sequence analysis suggests a great deal of similarity between the two. Despite this, however, mutational analysis revealed that the 5' boundary of the VP16 promoter extends to ca. -90. This region includes an Sp1 binding site at -46, CAAT box homology at -77, and "E box" (CACGTG) at -85. Mutational and deletional analyses demonstrate that the proximal Sp1 site plays little or no role in promoter strength; despite this it can be shown to bind Sp1 protein using DNA mobility shift assays. Like the VP5 promoter, the VP16 promoter also requires an initiator element at the cap site. The VP16 Inr element differs in sequence from that of the VP5 promoter, and its deletion or mutation has a significantly smaller effect on promoter strength. The difference between these two Inr elements was confirmed by our finding that the VP16 initiator element binds to the 65-kDa YY1 transcription factor, and the VP5 Inr element competes poorly for the binding between the VP16 element and infected cell proteins in comparative bandshift assays. While the VP16 Inr sequence is identical to that of several murine TATA-less promoters, the VP16 Inr requires a TATA box for measurable activity.