![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Journal of Virology, April 2006, p. 3541-3548, Vol. 80, No. 7
0022-538X/06/$08.00+0 doi:10.1128/JVI.80.7.3541-3548.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
and
Thomas Shenk*
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014
Received 19 September 2005/ Accepted 19 January 2006
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The 
240-kb HCMV genome encodes >200 open reading frames (ORFs). HCMV is an enveloped virus: its genome is encased within a capsid, and a protein layer called the tegument resides between the capsid and envelope. The tegument proteins (3, 22), which are unique to herpesviruses, are delivered to cells upon infection and can act before the onset of viral gene expression to help initiate a productive infection. Examples of tegument proteins include the UL69 protein, which blocks cell cycle progression in late G1 (14); the UL82-coded pp71, which acts as a transcriptional transactivator (10) and stimulates quiescent cells to enter the mitotic cycle (14); the UL83-coded pp65 and IRS1 plus TRS1 proteins, which antagonize aspects of the cellular antiviral response (1, 5, 6); and the UL99-coded pp28, which mediates the cytoplasmic envelopment of tegument capsids (20).
The UL26 protein (pUL26) is present in the tegument of the virion (3, 21, 22). It is expressed with early kinetics, and synthesis of the protein initiates at one of two start codons, yielding either a 21- or 27-kDa product. A segment of the UL26 coding region was isolated in an activator trap assay, arguing that the protein includes a transcriptional activator domain (21).
We analyzed the function of pUL26 by examining the phenotype of a UL26 deletion mutant. The mutant exhibited a strong growth defect in fibroblasts. Its virions contained normal levels of the tegument proteins that were assayed, but at least one of them, UL99-coded pp28, was hypophosphorylated. A reduced level of hypophosphorylated pp28 was evident in newly infected cells, suggesting that it is unstable, and there was a marked delay in the accumulation of IE1 mRNA and protein.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
HFFs expressing pUL26 were generated by infection with a recombinant retrovirus. To generate the retrovirus, the UL26 ORF was amplified from pAD/Cre (24), using the following primers: AGT TTG AGC GAA TTC ATG TAC GCC GTT TTC and ATT TTG CCC GGG CAA CAG CGC TGA TGG. The PCR-generated product was cloned into the Xma/EcoR1 sites of pBluescript, and two short DNA fragments (CCG GGG ACT ACA AAG ACC ATG ACG GTG ATT ACCA AGG ATG ACG ATG ACT AAGC and GGC CGC TTA GTC ATC GTC ATC CTT GTA ATC ACC GTC ATG GTC TTT GTA GTCC), containing two Flag-tag epitopes, were then cloned in-frame at the C terminus of the UL26 coding region to generate pBlueUL26F. The UL26-Flag fragment was subcloned from pBlueUL26F into pLPCX (Clontech), creating pLPCX-26F, and a retrovirus stock, retro-26F, was prepared by transfecting this plasmid into Phoenix Ampho cells (11). HFFs were infected with retro-26F to generate a population of FLAG-tagged pUL26 (pUL26F)-expressing cells.
To produce a UL26-specific monoclonal antibody, the UL26-Flag DNA segment from pBlueUL26F was subcloned into pGex4T1 to create pGex26F. A glutathione S-transferase (GST) fusion protein, GST-26F, was then produced by transfection of Escherichia coli BL-21 with pGex26F. The resulting fusion protein was affinity purified on glutathione Sepharose as recommended by the manufacturer (Amersham Pharmacia Biotech). Purified protein was used to immunize 5-week-old BALB/c mice. Mice were boosted with GST-26F twice and screened for seroconversion, and spleens were harvested for fusion. Hybridomas secreting antibody to pUL26 were screened, selected, and amplified. In addition to pUL26-specific antibody, monoclonal antibodies to the following viral proteins were employed: IRS1 (1B4 [17]), TRS1 (9A1 [17]) UL99-coded pp28 (10B4-29 [20]), UL83-coded pp65 (8F5 [16]), UL123-coded IE1 (1B12 [A. Marchini, P. Robinson, and T. Shenk, unpublished]), pUL44 (10D8; Virusys), and the UL86-coded major and UL85-coded minor capsid proteins (kind gifts from W. Gibson, Johns Hopkins).
Analysis of viral DNA, RNA, and protein. Viral DNA accumulation was monitored by slot blot assay. Cells were scraped, pelleted by low-speed centrifugation, washed with phosphate-buffered saline (PBS), and lysed in buffer containing 100 mM NaCl, 10 mM Tris (pH 8.0), 25 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), and 0.1 mg of proteinase K/ml. After incubation for 3 h at 55°C, DNA was extracted with phenol-chloroform and precipitated with ethanol. Aliquots (1 µg) were transferred to a nylon membrane using a slot blot apparatus. Immobilized viral DNA was hybridized to either a 32P-labeled random-primed probe from pAD/Cre DNA or a digoxigenin-labeled probe from a PCR-generated fragment of the UL36 gene generated using a digoxigenin probe synthesis kit (Roche).
For the analysis of IE1 mRNA, total RNA was prepared from infected cells, using Trizol as recommended by the manufacturer (Invitrogen). Equivalent amounts of RNA was then glyoxalated, subjected to 1.0% agarose gel electrophoresis, and transferred to a nylon membrane (2). Methylene blue staining of the membrane was used to ensure equivalent RNA loading and transfer. A 32P-labeled probe specific for IE1 was generated by random-primed synthesis from an IE1 plasmid (12). Northern hybridization was performed as described elsewhere (2).
Protein accumulation was assayed by Western blotting. Cells were scraped, pelleted by low-speed centrifugation, washed with PBS and solubilized in cellular lysis solution in buffer containing 150 mM KCl, 10 mM HEPES, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 10 mM NaF, and 100 µM sodium orthovanadate. After 10 min on wet ice, lysates were centrifuged at 14,000 x g for 10 min. The protein concentration of supernatant fluids was determined by using the Bio-Rad assay, according to the directions of the manufacturer. Equivalent amounts of protein (10 to 60 µg) were denatured in disruption buffer (50 mM Tris [pH 7.0], 2% SDS, 5% 2-mercaptoethanol, and 2.75% sucrose), subjected to electrophoresis in an SDS-containing 11% polyacrylamide gel, and transferred to a nitrocellulose sheet. Blots were then stained with Ponceau S to ensure equivalent protein loading and transfer, blocked by treatment with 5% milk, and reacted with primary antibody. Protein bands were visualized through enhanced chemiluminescent detection, according to the instructions of the manufacturer (Pierce).
To produce partially purified virions for the analysis of their constituent proteins, virus stocks were first clarified by low-speed centrifugation and then centrifuged through a sorbitol cushion at 55,000 x g for 1 h. The virion pellet was then resuspended in buffer containing 50 mM Tris, pH 7.4, and 100 mM NaCl and purified by centrifugation through a glycerol tartrate gradient as previously described (3). Bands containing virions were collected and virions were repelleted by centrifugation through a sorbitol cushion at 55,000 x g for 1 h and resuspended in either cellular lysis solution (described above), 2-D rehydration buffer (2% CHAPS, 8 M urea and 0.5% 3-10 IPG buffer) (Amersham Pharmacia Biotech) or 
phosphatase buffer (50 mM Tris [pH 7.5], 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brig 35, 2 mM MnCl2).
For two-dimensional (2-D) gel analysis of virion proteins, virions were resuspended in 250 µl of 2-D rehydration buffer, centrifuged to pellet insoluble material, loaded onto a 13-cm Immobiline Drystrep isoelectric-focusing gel (pH 7 to 10) (Amersham Biotech), and proteins were separated using the IPGphor isoelectric-focusing system as directed by the manufacturer (Amersham). After focusing, gels were equilibrated for 15 min in 50 mM Tris (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 10 mg/ml dithiothreitol, then the proteins in the gel were subjected to electrophoresis in the second dimension through an SDS-containing 11% polyacrylamide gel, and proteins were identified by Western blot assay, as described above.
For 2-D gel analysis of phosphatase-treated virions, partially purified virions were resuspended in phosphatase buffer with or without phosphatase inhibitors (10 mM NaF, 100 µM sodium orthovanadate). Virions were then incubated at 30°C with or without 800 units of protein phosphatase (New England Biolabs) for 1 h, then the sample volume was brought up to 250 µl with 2-D rehydration buffer and samples were processed for 2-D gel analysis as described above.
For immunofluorescence, fibroblasts were grown on glass coverslips. At various times postinfection, cells were washed twice with PBS, fixed with 2% paraformaldehyde in PBS for 20 min, washed twice with PBS, and permeablized with 0.1% TX-100 for 15 min when staining with pp28-specific antibody or permeabilized with 0.1% TX-100 and 0.1% SDS for 15 min when staining with pUL26-specific antibody. Cells were subsequently washed twice with PBS containing 0.05% Tween-20 and blocked by incubation for 1 h in PBS containing 2% bovine serum albumin and 0.05% Tween-20. Cells were incubated with primary antibody diluted in PBS plus 0.05% Tween-20 for 1 h, washed in the same buffer lacking antibody three times, incubated with fluorochrome-conjugated anti-mouse secondary antibody and DAPI (4',6'-diamidino-2-phenylindole) as a nuclear stain for 1 h, and washed again three times. Coverslips were mounted in slow-fade solution (Molecular Probes) and analyzed by confocal microscopy.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
Deletion of the UL26 ORF delays the onset of immediate early gene expression. To determine whether the BADsubUL26 defect resides before or after the initiation of viral DNA replication, the kinetics of viral DNA accumulation was monitored. After infection of fibroblasts at a multiplicity of 3 PFU/cell, the mutant and wild-type viruses accumulated viral DNA similarly (Fig. 3D, left panel). In contrast, after infection at a lower input multiplicity (0.1 PFU/cell), the accumulation of BADsubUL26 DNA was delayed (Fig. 3D, right panel). An increase over input levels of DNA was evident for the wild-type virus at 72 h postinfection, whereas the increase was not evident for BADsubUL26 DNA until 120 h postinfection. Thus, the more modest defect observed after infection at a higher input multiplicity does not appear to strongly affect the kinetics of viral DNA replication, whereas the defect observed after infection at 0.1 PFU/cell negatively impacts infection either at or prior to DNA replication. These results support our finding that the mutant exhibits a greater growth defect after infection at a relatively low than at a higher input multiplicity (Fig. 3A).
To more precisely identify the step during the viral life cycle compromised by the mutation in BADsubUL26, infected fibroblasts were assayed by Western blot for the accumulation of several viral proteins (Fig. 4A). The mutant exhibited a transient delay in the accumulation of several immediate early proteins. IE1 protein was present at a markedly reduced level at 8 h, but by 24 h, it had accumulated to wild-type levels. Immediate-early pIRS1 was detected in BADwt, not in BADsubUL26 lysates at 8 h postinfection. It accumulated to a reduced level in mutant infected cells at 24 h, and reached levels comparable to the wild type at 48 h postinfection. There was also significantly less immediate-early pTRS1 at 24 h in BADsubUL26 lysates compared to the wild type, whereas at 48 h, the levels were roughly equivalent. At 24 h, BADsubUL26 also accumulated slightly less pUL44, an early protein, yet by 48 h, levels of UL44 protein accumulation in mutant and wild-type virus infections were similar.
|
Deletion of the UL26 ORF results in reduced intracellular levels of several tegument proteins delivered in virions. Interestingly, there seemed to be less pp28 and pp65 present in cells at 8 h after infection with BADsubUL26 compared to the wild-type virus (Fig. 4A). The levels of pp28 and pp65 in mutant-infected fibroblasts appeared to reach wild-type levels by 24 and 48 h, respectively (Fig. 4A). Since both pp28 and pp65 are late proteins, their levels at 8 h should result from their delivery to newly infected cells as components of the virion's tegument. The reduced levels of these proteins at 8 h could result from a defect in the packaging of pp28 and pp65 into infectious ADsubUL26 particles, inefficient adsorption/entry of mutant virions into cells, or reduced stability of pp28 and pp65 once they have been delivered to the newly infected cells.
We first tested the possibility that less pp28 and pp65 was packaged into BADsubUL26 particles. Mutant and wild-type virions were purified by centrifugation in a glycerol tartrate gradient, which separates infectious virions from noninfectious virus particles (4). Virion bands were removed from the gradient and normalized for total protein content, and selected virion proteins were analyzed by Western blotting (Fig. 5A). The minor capsid protein was present in similar amounts in the mutant and wild-type virion preparations, which argues that roughly equivalent numbers of mutant and wild-type virions were assayed in the experiment. The pp28 and pp65 phosphoproteins were present in the same proportion as the minor capsid protein in mutant compared to wild-type virions, which argues that they are packaged in normal quantities in the absence of pUL26.
|
We next tested the levels of cell-associated pp65 and pp28 very early after infection of fibroblasts with mutant or wild-type virus. Adsorption was allowed to proceed for 30 min at a multiplicity of 3 PFU/cell, and cells were washed with sodium citrate to inactivate virus on the cell surface and harvested at various times thereafter. At 30 min, there was a significant decrease in the amounts of pp28 and pp65 present in cells infected with BADsubUL26 compared to ADwt (Fig. 5C).
Mutant virions contain normal levels of the proteins (Fig. 5A) and adsorption proceeds with normal kinetics (Fig. 5B), but reduced levels of the pp28 and pp65 virion proteins are present in the cell during the early stages of infection (Fig. 5C and D). Do the reduced intracellular levels of virion proteins result from inefficient fusion and delivery of the proteins into cells or from rapid degradation of the proteins within the cells? A normal amount of viral DNA (Fig. 5B) but also reduced quantities of virion proteins (Fig. 5C) become cell associated at 30 min and 2 h after infection with the mutant. Since a fusion/entry defect would concomitantly affect the intracellular levels of both DNA and proteins, we can conclude that fusion/entry is normal and a portion of the mutant proteins is rapidly degraded after infection.
Since a portion of several BADsubUL26 tegument proteins was quickly degraded after infection, it was of interest to determine if these proteins were properly localized during the early stages of infection. The localizations of pp65 and pp28 as assayed by immunofluorescence at 4 h postinfection were not discernibly different between ADwt and BADsubUL26 (Fig. 5D), suggesting that gross localization changes are not responsible for their degradation.
BADsubUL26 virions contain hypophosphorylated pp28. Although pp28 is present at roughly normal levels in BADsubUL26 virions (Fig. 5A), a portion of the phosphoprotein is rapidly degraded after the tegument-associated capsid enters the newly infected cell (Fig. 5C and D). It seemed possible that the degradation could result from abnormal phosphorylation of pp28. In some but not all experiments, the pp28 delivered to cells by mutant virions appeared to migrate more rapidly than the pp28 delivered by wild-type virions (Fig. 5C), as might occur if the protein were hypophosphorylated. To explore this possibility, the proteins in gradient-purified BADsubUL26 and ADwt virions were separated by 2-D gel electrophoresis and pp28 was identified in the patterns by Western blot assay. Multiple pp28 protein isoforms were present in wild-type virions, and several of these isoforms were present at substantially reduced levels in mutant virions (Fig. 6A), indicating that pUL26 is needed for proper posttranslational modification of virion pp28. The pp28 isoforms that were underrepresented in BADsubUL26 were more slowly migrating and more acidic, suggesting that the pp28 in mutant virions is hypophosphorylated. This interpretation was confirmed by treating ADwt virions with phosphatase (Fig. 6B). This treatment eliminated the pp28 isoforms from the wild-type virion lysates that were underrepresented in the mutant virion lysates. These results demonstrate that pUL26 protein is required for the assembly of virions with properly phosphorylated pp28. Hypophosphorylated pp28 can be encapsidated in virions, but the protein is unstable when it is released into newly infected cells.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A pUL26-deficient virus, BADsubUL26, exhibited a multiplicity-dependent growth defect in fibroblasts (Fig. 3A). The mutant exhibited a significant kinetic delay after infection at a relatively low multiplicity (0.01 PFU/cell); the delay was not evident after infection at a higher multiplicity (3 PFU/cell). The mutant spread very slowly from cell to cell, taking much longer to form plaques (1 month compared to 2 weeks for wild-type virus [data not shown]). BADsubUL26 virions exhibited a normal PFU/particle ratio. BADsubUL26 and ADwt virion samples with equal amounts of infectivity contained the same amount of viral DNA and delivered the same amount of viral DNA to newly infected cells (Fig. 5B). A virus in which the mutation was repaired, BADrevUL26, grew like wild-type virus (Fig. 3A) and pUL26 supplied in trans partially rescued the mutant's growth defect (Fig. 3C), arguing that the poor growth exhibited by the mutant virus results from the loss of pUL26 function.
The earliest consequence that we observed of a pUL26 deficiency during infection involved incoming virion proteins. Two virion tegument phosphoproteins, pp28 and pp65, accumulated to reduced levels within mutant-virus-infected fibroblasts (Fig. 5C). We initially suspected that BADsubUL26 virions might contain reduced levels of the two tegument proteins, but they proved to package normal quantities of pp28 and pp65 (Fig. 5A). Adsorption was found to progress with normal kinetics by monitoring the association of viral DNA with cells (Fig. 5A). Fusion and entry were judged to be normal due to the discordant relative levels of cell-associated viral DNA and tegument proteins in mutant compared to wild-type-virus-infected cells. A defect in fusion and entry would affect internalization of all virion components equally. However, the amount of cell-associated DNA was normal (Fig. 5B), whereas the levels of pp28 and pp65 tegument phosphoproteins were reduced at 30 min after infection with BADsubUL26 compared to ADwt (Fig. 5C).
Importantly, even though BADsubUL26 virions contain normal amounts of pp28 (Fig. 5A), a portion of the pp28 in mutant virions is hypophosphorylated (Fig. 6). We have not yet tested the phosphorylation states of additional tegument proteins, but others might well be affected, since pp65, like pp28, is rapidly degraded after (Fig. 5C). This leads us to propose that abnormal phosphorylation leads to the rapid degradation of pp28 and likely additional tegument proteins after BADsubUL26 infection. Conceivably, the change in phosphorylation might alter the normal disassembly of the tegument, and the abnormal disassembly might lead to extensive degradation of its constituent proteins. Alternatively, hypophosphorylation might alter the interaction of pp28 with other proteins in the newly infected cell, and its failure to interact properly would lead to its destruction. To test the possibility that the proteasome mediates the degradation of a portion of the input pp28 at the start of BADsubUL26 infection, fibroblasts were infected in the presence or absence of the proteasome inhibitor, MG132. The drug did not affect the levels of pp28 present after infection with mutant virus compared to wild-type virus (data not shown) suggesting that the excessive degradation of BADsubUL26 virion-derived pp28 is not proteasome dependent.
Given the hypophosphorylation of pp28 in BADsubUL26 virions, it is very likely that pUL26 influences the phosphorylation state of some and perhaps all HCMV tegument proteins. It might serve to direct the proper phosphorylation of tegument proteins as they are made during the late phase of infection. It is conceivable that pUL26 acts indirectly, perhaps by influencing the expression of a cellular kinase or phosphatase at the level of transcription. This notion receives support from the fact that pUL26 is localized to the nucleus during the early phase of HCMV infection (Fig. 2A and B), and earlier work has demonstrated that it contains a transcriptional activation domain (21). Alternatively, pUL26 could act directly to influence the phosphorylation of tegument proteins. It does not have a kinase active-site motif, a phosphatase active-site motif, or an ATP-binding site that we can recognize, but pUL26 could bind to a viral or cellular kinase and redirect its activity to tegument proteins, or it might bind to a phosphatase and prevent it from acting on tegument proteins. The localization of pUL26 to the juxtanuclear virion assembly domain late after infection (Fig. 2C to E) positions it to perform either of these postulated activities. It also remains possible that pUL26 does not influence the phosphorylation of tegument proteins but normally serves a gating function, excluding the encapsidation of hypophosphorylated proteins.
HCMV encodes a known protein kinase, pUL97, but we were unable to detect an interaction with pUL26 and we did not observe a change in its location after infection with the pUL26-deficient mutant (data not shown). We are currently searching for interacting cellular proteins.
Since purified BADsubUL26 virions contain hypophosphorylated pp28 (Fig. 6), pUL26 must function during the late phase of infection. The timing of its action might explain the partial complementation of BADsubUL26 replication by pUL26 provided in trans (Fig. 3C). If pUL26 normally controls phosphorylation of tegument proteins or gates their incorporation into virions during the late phase of infection, it might not effectively prevent the degradation of incoming hypophosphorylated proteins at the start of infection.
The earliest defect in the program of HCMV gene expression that we observed after infection with BADsubUL26 was a marked delay in the accumulation of several immediate-early proteins: IE1, pIRS1, and pTRS1 (Fig. 4). An effect on immediate-early expression is again consistent with our observation that pUL26 localizes to the nucleus early during infection (Fig. 2A and B) and with a previous report that domain within pUL26 can upregulate major immediate-early promoter activity in transient transfection assays (21). However, pUL26 could well act indirectly to influence expression of the major immediate-early gene product, given our demonstration that it stabilizes the pp28 and pp65 tegument phosphoproteins (Fig. 4A and 5C). For example, pUL26 might also stabilize the UL82-coded pp71 tegument phosphoprotein, which activates transcription in transfection assays (13) and enhances the accumulation of several immediate-early mRNAs within infected cells (4).
We nevertheless tested the possibility that reduced IE1 expression is responsible for the BADsubUL26 growth defect. Since the inhibition of histone deacetylase (HDAC) activity can rescue the growth of an IE1-null mutant (15), we reasoned that treatment with an HDAC inhibitor would rescue BADsubUL26 replication if its replication defect was ultimately due to reduced IE1 expression. However, trichostatin A, an HDAC inhibitor that compensates for the loss of IE1 activity, had no effect on BADsubUL26 replication (data not shown). Not surprisingly, then, an effect on IE1 activity is not solely responsible for the BADsubUL26 replication defect.
In conclusion, a pUL26-deficient mutant produces virions with hypophosphorylated pp28 protein, and a portion of this virion protein is rapidly degraded at the start of infection. We propose that pUL26 regulates the phosphorylation of several if not all tegument proteins, and we speculate that proper phosphorylation is necessary for their normal function and stability.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by a grant from the NIH (CA82396), and J.M. was supported by a postdoctoral fellowship from the American Cancer Society.
|
FOOTNOTES |
|---|
Present address: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | Mol. Cell. Biol. | Microbiol. Mol. Biol. Rev. |
|---|
| Clin. Vaccine Immunol. | ALL ASM JOURNALS |
|---|
), BADsubUL26 (
), or BADrevUL26 (
). Cells were harvested at the indicated times, and infectious viral progeny was quantified by plaque assay on fibroblasts. (B) Mock-infected fibroblasts transduced with a retrovirus expressing pUL26 as well as mock- or ADwt-infected (multiplicity of 3.0 PFU/cell) nontransduced fibroblasts were harvested and processed for Western blotting using a pUL26-specific monoclonal antibody. (C) Production of infectious progeny in fibroblasts expressing pUL26. Replicate cultures of retrovirally transduced fibroblasts expressing pUL26 were infected at a multiplicity of 0.01 PFU/cell with ADwt (
