Important role for the murid herpesvirus 4 ribonucleotide reductase large subunit in host colonization via the respiratory tract.
ABSTRACT Viral enzymes that process small molecules provide potential chemotherapeutic targets. A key constraint-the replicative potential of spontaneous enzyme mutants-has been hard to define with human gammaherpesviruses because of their narrow species tropisms. Here, we disrupted the murid herpesvirus 4 (MuHV-4) ORF61, which encodes its ribonucleotide reductase (RNR) large subunit. Mutant viruses showed delayed in vitro lytic replication, failed to establish infection via the upper respiratory tract, and replicated to only a very limited extent in the lower respiratory tract without reaching lymphoid tissue. RNR could therefore provide a good target for gammaherpesvirus chemotherapy.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: We studied the in vivo biological properties of viruses reconstituted from the genome of murine gammaherpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus RgammaHV68A98.01, containing BAC vector sequences, is attenuated in vivo as determined by (i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen cells reactivating virus in an ex vivo reactivation assay. Since the BAC vector sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus RgammaHV68A98.02, with biological properties comparable to those of wild-type MHV-68. On the basis of these data we conclude (i) that excision of BAC vector sequences from cloned MHV-68 genomes is critical for reconstitution of the wild-type phenotypic properties of this virus and (ii) that the BAC-cloned MHV-68 genome is suitable for the construction of mutants and mutant libraries whose phenotypes can be reliably assessed in vivo.Journal of Virology 07/2001; 75(12):5692-6. · 5.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Human cytomegalovirus infects vascular tissues and has been associated with atherogenesis and coronary restenosis. Although established laboratory strains of human cytomegalovirus have lost the ability to grow on vascular endothelial cells, laboratory strains of murine cytomegalovirus retain this ability. With the use of a forward-genetic procedure involving random transposon mutagenesis and rapid phenotypic screening, we identified a murine cytomegalovirus gene governing endothelial cell tropism. This gene, M45, shares sequence homology to ribonucleotide reductase genes. Endothelial cells infected with M45-mutant viruses rapidly undergo apoptosis, suggesting that a viral strategy to evade destruction by cellular apoptosis is indispensable for viral growth in endothelial cells.Science 02/2001; 291(5502):303-5. · 31.03 Impact Factor
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ABSTRACT: All gammaherpesviruses encode a virion glycoprotein positionally homologous to Epstein-Barr virus gp350. These glycoproteins are thought to be involved in cell binding, but little is known of the roles they might play in the whole viral replication cycle. We have analyzed the contribution of murine gammaherpesvirus 68 (MHV-68) gp150 to viral propagation in vitro and host colonization in vivo. MHV-68 lacking gp150 was viable and showed normal binding to fibroblasts and normal single-cycle lytic replication. Its capacity to infect glycosaminoglycan (GAG)-deficient CHO-K1 cells and NS0 and RAW264.7 cells, which express only low levels of GAGs, was paradoxically increased. However, gp150-deficient MHV-68 spread poorly through fibroblast monolayers, with reduced cell-free infectivity, consistent with a deficit in virus release. Electron microscopy showed gp150-deficient virions clustered on infected-cell plasma membranes. MHV-68-infected cells showed reduced surface GAG expression, suggesting that gp150 prevented virions from rebinding to infected cells after release by making MHV-68 infection GAG dependent. Surprisingly, gp150-deficient viruses showed only a transient lag in lytic replication in vivo and established normal levels of latency. Cell-to-cell virus spread and the proliferation of latently infected cells, for which gp150 was dispensable, therefore appeared to be the major route of virus propagation in an infected host.Journal of Virology 06/2004; 78(10):5103-12. · 5.08 Impact Factor
JOURNAL OF VIROLOGY, Oct. 2010, p. 10937–10942
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 20
Important Role for the Murid Herpesvirus 4 Ribonucleotide
Reductase Large Subunit in Host Colonization
via the Respiratory Tract?
Michael B. Gill, Janet S. May, Susanna Colaco, and Philip G. Stevenson*
Division of Virology, Department of Pathology, University of Cambridge, Cambridge, United Kingdom
Received 19 April 2010/Accepted 8 July 2010
Viral enzymes that process small molecules provide potential chemotherapeutic targets. A key constraint—
the replicative potential of spontaneous enzyme mutants—has been hard to define with human gammaher-
pesviruses because of their narrow species tropisms. Here, we disrupted the murid herpesvirus 4 (MuHV-4)
ORF61, which encodes its ribonucleotide reductase (RNR) large subunit. Mutant viruses showed delayed in
vitro lytic replication, failed to establish infection via the upper respiratory tract, and replicated to only a very
limited extent in the lower respiratory tract without reaching lymphoid tissue. RNR could therefore provide a
good target for gammaherpesvirus chemotherapy.
Cellular deoxyribonucleotide synthesis is strongly cell cycle
dependent. DNA viruses replicating in noncycling cells must
therefore either induce cellular enzymes or supply their own.
Most herpesviruses encode multiple homologs of nucleotide
metabolism enzymes, including both subunits of the cellular
ribonucleotide reductase (RNR) (4). While most in vivo cells
are resting, most in vitro cell lines divide continuously (29). The
importance of viral RNRs may therefore only be apparent in
vivo (14). In contrast to alpha- and betaherpesviruses, gamma-
herpesviruses cause disease mainly through latency-associated
cell proliferation. However, gamma-2 herpesviruses show lytic
gene expression in sites of latency (9, 17), and lytic reactivation
could potentially alleviate some gammaherpesvirus-infected
cancers (7, 8). Therefore, it is important also to understand the
pathogenetic roles of gammaherpesvirus lytic cycle enzymes,
such as RNR.
The known human gammaherpesviruses Epstein-Barr virus
(EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV)
have narrow species tropisms that preclude most pathogenesis
studies. In contrast, murid herpesvirus 4 (MuHV-4) (21, 26)
allows gammaherpesvirus host colonization to be studied in
vivo. After intranasal (i.n.) inoculation, MuHV-4 replicates
lytically in lung epithelial cells before seeding to lymphoid
tissue (27). Long-term virus loads are independent of extensive
primary lytic spread (25). However, whether persistence re-
quires some lytic gene expression remains unclear. Replica-
tion-deficient viral DNA reached the spleen after intraper-
itoneal (i.p.) but not i.n. virus inoculation (15, 20, 28),
suggesting that virus dissemination from the lung to lym-
phoid tissue requires lytic replication. In addition, less in-
vasive inoculations may increase further the viral functions
required to establish a persistent infection. Thymidine ki-
nase (TK)-deficient MuHV-4 given i.n. without general an-
esthesia, in which method the wild-type virus infects the
upper respiratory tract and reaches lymphoid tissue without
infecting the lungs (18), fails to colonize in mice at all (12).
The implication is that virions using a likely physiological route
of host entry must replicate in terminally differentiated cells to
establish a significant infection. However, some unusual fea-
tures of gammaherpesvirus TKs (11) suggest that they have
functions besides thymidine phosphorylation. We therefore
targeted here another enzyme linked to viral DNA replication,
the MuHV-4 RNR. We aimed to define the in vivo importance
of a potential therapeutic target and to advance generally our
understanding of gammaherpesvirus pathogenesis.
Transposon insertions in the MuHV-4 RNR small (ORF60)
and large (ORF61) RNR subunit genes have been described as
either attenuating or not for lytic replication in vitro (19, 23).
We disrupted ORF61 (RNR?) by inserting stop codons close
to its 5? end (Fig. 1a). An EcoRI-L genomic clone (coordinates
80644 to 84996) in pUC19 (6) was digested with AleI to re-
move nucleotides 82320 to 82534 of ORF61 (82865 to 80514).
An oligonucleotide encoding multiple stop codons and an
EcoRI restriction site (5?-CTAGCATGCTAGAATTCTAGC
ATGCATG-3?) was ligated in place. Nucleotides 81365 to
83883 were then PCR amplified, including a BamHI site in the
81365 primer, cloned as a BglII/BamHI fragment into the
BamHI site of pST76K-SR, and recombined into a MuHV-4
bacterial artificial chromosome (BAC) (1). A revertant virus
was made by reconstituting the corresponding, unmutated
genomic fragment. Southern blots (5) of viral DNA (Fig. 1b)
confirmed the expected genomic structures, and immunoblots
(5) of infected cell lysates (Fig. 1c) established that mutant
viruses no longer expressed the RNR large subunit.
RNR?viruses were noticeably slower than RNR?viruses
when spreading through BHK-21 cell monolayers after BAC
DNA transfection. Normalizing by immunoblot signal, RNR?
virus stocks had titers similar to that of the wild type by viral
enhanced green fluorescent protein (eGFP) expression but 10- to
100-fold lower plaque titers. Using eGFP expression as a readout,
RNR?virion production after a low multiplicity of infection
lagged 1 day behind that of the wild type (Fig. 1d). Maximum
* Corresponding author. Mailing address: Division of Virology,
Department of Pathology, Tennis Court Road, Cambridge CB2
1QP, United Kingdom. Phone: 44-1223-336921. Fax: 44-1223-
336926. E-mail: firstname.lastname@example.org.
?Published ahead of print on 28 July 2010.
infectivity yields were also reduced, but once BHK-21 cells be-
come confluent, they support MuHV-4 lytic infection poorly, so
this was probably a consequence of the slower lytic spread. After
a high multiplicity of infection (Fig. 1e), RNR?mutants showed
a 10-h lag in virion production and no difference in the final yield.
They showed no defect in single-cycle eGFP expression (Fig. 1f),
implying normal virion entry. Therefore, the main RNR?defect
lay in infectious virion production.
FIG. 1. Disruption of the MuHV-4 ORF61. (a) Schematic diagram of the ORF61 (RNR large) locus, showing the mutation introduced and
relevant restriction sites. (b) Viral DNA was digested with EcoRI and probed for ORF61. Oligonucleotide insertion into ORF61 changes a
4,352-bp wild-type band to 2,462 bp plus 1,676 bp. The 2,462-bp fragment is not visible because it overlaps the probe by only 331 nucleotides (nt)
and comigrates with a background band of unknown origin. WT, wild type; REV, revertant; RNR?, mutant; RNR?ind, independent mutant. WT
luc?is MuHV-4 expressing luciferase from an ORF57/ORF58 intergenic cassette. RNR?luc?and RNR?luc?ind have ORF61 disrupted on this
background. (c) Infected cell lysates were immunoblotted for gp150 (virion envelope glycoprotein, monoclonal antibody [MAb] T1A1), ORF17
(capsid component, MAb 150-7D1), TK (tegument component, MAb CS-4A5), and ORF61 (MAb PS-8A7). (d) BHK-21 cells were infected with
RNR?or RNR?viruses (0.01 eGFP units/cell, 2 h, 37°C), washed two times with phosphate-buffered saline (PBS) to remove unbound virions, and
cultured at 37°C to allow virus spread. Infectivity (in eGFP units) at each time point was determined on fresh BHK-21 cells in the presence of
phosphonoacetic acid to prevent further viral spread, with the number of eGFP-postive cells counted 18 h later by flow cytometry. (e) BHK-21 cells
were infected with RNR?or RNR?viruses (2 eGFP units/cell, 2 h, 37°C), washed in medium (pH 3) to inactivate nonendocytosed virions, and
cultured at 37°C to allow virus replication. The infectivity of replicate cultures was then assayed as described in the legend of panel d. (f) BHK-21
cells were incubated with RNR?or RNR?viruses (0.3 eGFP units/cell, 37°C) for the times indicated, and the numbers of eGFP-positive cells in
the cultures were then determined by flow cytometry.
10938 NOTESJ. VIROL.
FIG. 2. Host colonization by RNR?MuHV-4 mutants. (a) BHK-21 cells were left uninfected or infected overnight with RNR?or RNR?luc?
MuHV-4 and then assayed for luciferase expression by luminometry. Phosphonoacetic acid (PAA; 100 ?g/ml) was either added or not to cultures
to block viral late gene expression. Each point shows the mean ? standard deviation from triplicate cultures. (b) BALB/c mice were infected i.n.
under general anesthesia with RNR?or RNR?luc?MuHV-4 (5 ? 103PFU) and then assayed for luciferase expression by luciferin injection and
CCD camera scanning. The images are from 5 days postinfection. Note that the RNR?and RNR?images have different sensitivity scales. (c) For
quantitation, dorsal and ventral luciferase signals were summed. Each point shows 1 mouse. The dashed lines show detection thresholds. The
RNR?signal was significantly greater than the RNR?signal for all sites and time points (P ? 0.001 by Student’s t test). (d) C57BL/6 mice were
infected i.n. under anesthesia with RNR?or RNR?MuHV-4 (5 ? 103PFU). Five days later, infectious virus loads in noses and lungs were
measured by plaque assay. Each point shows 1 mouse. RNR?infections yielded no plaques and therefore are shown at the sensitivity limits of each
assay. (e) BALB/c mice were infected i.n. with RNR?or RNR?MuHV-4 without anesthesia and then monitored by luciferin injection and CCD
camera scanning. Each point shows the summed ventral and dorsal signals of the relevant region for 1 mouse. Neck signals correspond to the
superficial cervical lymph nodes (SCLN). The dashed lines show detection thresholds. RNR?luciferase signals were undetectable at all time points.
VOL. 84, 2010NOTES 10939
For in vivo experiments, the loxP-flanked viral BAC-eGFP
cassette must be removed (1). Therefore, to monitor infection
in vivo without having to rely on new virion production as a
readout, we transferred the RNR mutation onto a luciferase-
positive (luc?) background (18). Viral luciferase expression
(from an early lytic promoter) by in vitro luminometry (18) was
independent of either viral DNA replication or RNR expres-
sion (Fig. 2a). After i.n. inoculation of anesthetized mice,
RNR?luciferase signals measured in vivo by i.p. luciferin in-
jection and IVIS Lumina charge-coupled-device (CCD) cam-
era scanning (18) were visible in lungs (Fig. 2b) but were
100-fold lower than those of the RNR?controls (Fig. 2c). A
severe impairment of RNR?lytic replication was confirmed by
plaque assay (18) (Fig. 2d); the difference between RNR?and
RNR?plaque titers greatly exceeded any difference in plaqu-
No RNR?luciferase signals were visible in noses, nor did
RNR?MuHV-4 give signals in the superficial cervical lymph
nodes (SCLN), which drain the nose (Fig. 2c). This lack of live
imaging signals from the upper respiratory tract was confirmed
by ex vivo imaging of SCLN at day 14 postinfection. We exam-
ined upper respiratory tract infection further with an indepen-
dently derived luc?RNR?mutant, inoculating i.n. without
anesthesia so as to avoid virus aspiration into the lungs. No
RNR?luciferase signals were detected, while wild-type signals
were readily observed in the nose and superficial cervical
lymph nodes (Fig. 2e).
Like RNR?MuHV-4, TK?mutants are severely attenuated
for lytic replication in the lower respiratory tract. However,
they eventually establish a reactivatable latent infection and
induce virus-specific antibody (3). Latent virus titers in spleens
peak at 1 month postinoculation. Infectious center assays
showed no RNR?infection of spleens at that time (Fig. 3a).
We also looked for viral DNA in spleens by quantitative PCR
(Fig. 3b). Genomic coordinates 4166 to 4252 were amplified
and hybridized to a probe with coordinates 4218 to 4189. Viral
genome copies, relative to the cellular adenosine phosphori-
bosyl transferase copy number, were calculated from standard
FIG. 3. Spleen colonization by RNR?MuHV-4. (a) BALB/c or C57BL/6 mice were infected i.n. either with general anesthesia (lung infection)
or without (nose infection). One month later, spleens were assayed for recoverable latent virus by infectious center assay. Lower detection limit,
10 infectious centers per spleen. (b) The spleens described in the legend of panel a were further analyzed for viral DNA by quantitative PCR. Copy
numbers are expressed relative to the cellular adenosine phosphoribosyl transferase copy number in each sample. The dashed lines show lower
detection limits (1 viral copy/10,000 cellular copies). (c) Sera from BALB/c mice after i.n. infection either with (lung infection) or without (nose
infection) general anesthesia were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance curve for 1 mouse. The dashed
lines show naive serum. (d) Sera from C57BL/6 mice 1 month after infection with independent RNR mutants were analyzed for MuHV-4-specific
IgG, as described in the legend to panel c.
curves of cloned plasmid DNA (10). No RNR?viral DNA was
detected. ELISA for MuHV-4-specific serum IgG (24) de-
tected an antibody response after lung infection but not upper
respiratory tract infection of BALB/c mice with RNR?
MuHV-4 (Fig. 3c). There was a similar lack of antibody 1
month after upper respiratory tract infection of C57BL/6
mice with independently derived RNR?mutants (Fig. 3d)
and 3 months after exposure of 6 BALB/c mice to RNR?
luc?MuHV-4. In contrast, i.p. RNR?luc?MuHV-4 gave
lower luciferase signals than RNR?luc?MuHV-4 (Fig. 4a),
but RNR?infectious centers (Fig. 4b) and viral genomes
(Fig. 4c) were detected in spleens, and enzyme-linked im-
munosorbent assays (ELISAs) (Fig. 4d) showed MuHV-4-
specific serum IgG.
The failure of both the RNR large subunit (ORF61) and
TK?MuHV-4 mutants to infect via the upper respiratory tract
argues that this requires viral replication in a nucleotide-poor
cell. The additional lack of lymphoid RNR?infection after
inoculation into the lungs seemed likely to reflect a defect in
virus transport, as RNR?MuHV-4 did colonize the spleen
after i.p. inoculation. It is also possible that the first cells
infected simply produced no infectious virions, although this
seemed a more likely explanation for upper respiratory tract
infection being undetectable; lung infection progressed suffi-
ciently to give detectable luciferase expression and to induce
an antiviral antibody response. How transport from lung to
lymphoid tissue occurs is unknown, but likely scenarios include
latently infected dendritic cells (22) carrying MuHV-4 along
afferent lymphatics to germinal centers and cell-free virions
being captured in lymph nodes by subcapsular sinus macro-
phages (13). Therefore, RNR may be important for MuHV-4
to spread from myeloid cells to B cells.
The difference between RNR?and TK?mutants in host
colonization via the lung—TK?mutants reached lymphoid
tissue whereas RNR?mutants did not—could reflect addi-
tional ORF61 functions, as precedent exists for functional drift
(2, 16). Alternatively, RNR may be needed more than TK for
MuHV-4 replication in some cell types. Formidable hurdles to
RNR-based therapies remain: human gammaherpesvirus in-
fections rarely present until latency is well established, so
blocking virus spread to lymphoid tissue may have a limited
impact, and no drugs sufficiently selective to target viral RNRs
in a clinical setting have yet emerged. Nevertheless, the severe
in vivo attenuation of RNR?MuHV-4 suggested that RNR
may be a viable target for limiting gammaherpesvirus lytic
P.G.S. is a Wellcome Trust Senior Clinical Fellow (GR076956MA).
This work was also supported by Medical Research Council grant
G0701185 and by Wellcome Trust project grant WT089111MA.
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2. Brune, W., C. Me ´nard, J. Heesemann, and U. H. Koszinowski. 2001. A
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3. Coleman, H. M., de B. Lima, V. Morton, and P. G. Stevenson. 2003. Murine
gammaherpesvirus 68 lacking thymidine kinase shows severe attenuation of
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FIG. 4. Intraperitoneal infection with RNR?and RNR?MuHV-4. (a)
Mice were infected i.p. with RNR?luc?or RNR?luc?MuHV-4 and then
monitored for luciferase expression. Each point shows the total abdominal
background. (b) Spleens were assayed for recoverable virus by infectious
center assay 10 days after i.p. infection with RNR?luc?or RNR?luc?
MuHV-4. Each point shows the titer of 1 mouse. One log10infectious center
per mouse corresponds to the lower limit of detection. (c) Spleen DNA was
analyzed for viral genome content by quantitative PCR. Each point shows
viral copy/cellular copy for the mean of triplicate reactions for 1 mouse. (d)
Sera taken 10 days after i.p. infection with RNR?luc?or RNR?luc?
the absorbance values for the serum of 1 mouse. “Naive” represents age-
matched, uninfected controls.
VOL. 84, 2010NOTES10941