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Journal of Virology, September 2006, p. 8371-8378, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00399-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle

Xuyan Feng, Jörg Schröer, Dong Yu, and Thomas Shenk^*

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014

Received 24 February 2006/ Accepted 20 June 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human cytomegalovirus (HCMV) is a ubiquitous human pathogen and a member of the ß-herpesvirus family. Although the virus generally does not cause disease in healthy individuals, it can cause life-threatening disease in immunologically immature or compromised individuals, including neonates, AIDS patients, allogeneic-transplant recipients, and cancer patients undergoing chemotherapy (20, 25, 29).

The >230-kbp HCMV genome contains 12 members of the so-called US22 gene family (11, 27): UL23, UL24, UL28, UL29, UL36, UL43, IRS1, TRS1, US22, US23, US24, and US26. The US22 genes were originally grouped on the basis of their sequence relatedness, and all ß-herpesviruses contain family members. US22 proteins contain segments of hydrophobic and charged residues as well as up to four conserved sequence motifs (16). The conserved sequence motifs suggest a common ancestry and possibly related functions for these gene products. More recently, a pattern discovery-based multiple sequence alignment algorithm was used to demonstrate that four members of the family (US22, US23, US24, and US26) are significantly more closely related to each other than other members of the group (32). None of the US22 genes is essential for growth of the AD169 (46) or Towne (14) laboratory strain of HCMV in fibroblasts. UL23, UL24, UL36, UL43, IRS1, and US22 are completely dispensable for AD169 growth in these cells; indeed, the AD169 UL36 open reading frame (ORF) has suffered point mutations, and its encoded protein localizes improperly within infected cells (30), and UL43 is truncated in some AD169 strains (13, 26). UL28, UL29, TRS1, US23, US24, and US26 are augmenting genes, i.e., they enhance the yield of AD169 in fibroblasts (46).

Among the 12 HCMV US22 genes, UL36, TRS1, and IRS1 have been investigated in some detail. The UL36 protein was found to be a nonessential (30) cell death suppressor, which inhibits Fas-mediated apoptosis by binding to the prodomain of caspase-8 and preventing its activation (40). TRS1 and IRS1 proteins are packaged in virions (34) and can activate transcription in cooperation with other immediate-early gene products (42, 19, 33). An HCMV mutant lacking the TRS1 coding region exhibits a late defect (6), because the protein is required for efficient assembly of DNA-containing capsids (2). TRS1 as well as IRS1 was shown to complement the function of vaccinia virus E3L protein by blocking cellular antiviral responses (12). TRS1 was further shown to have double-stranded-RNA binding activity (15). Little is known about the functions of the other HCMV US22 gene family members, although most have been identified as encoding virion proteins (4, 34, 1, 44). No functional analysis of the closely related US22, US23, US24, and US26 genes has been reported.

Murine cytomegalovirus also contains 12 US22 family members (31), and viruses with mutations in each of the family members have been isolated (17, 24). Two of the murine family members, m142 and m143, are essential for replication in cultured fibroblasts, and the remainder are not required. Five family members, M36, M43, m139, m140, and m141, influence the host range of the virus and were required for efficient replication in macrophages. The proteins encoded by m139, m140, and m141 interact (24, 22).

In this report, we investigate the function of the HCMV US24 member of the US22 gene family. Earlier work has shown that US24 RNA is expressed with early kinetics (10) and that the US24 protein (pUS24) is incorporated into virions (44). We describe the characterization of two US24 mutants of AD169, which produce reduced yields of infectious progeny after infection at both high and low input multiplicities. When fibroblasts were infected with an equivalent number of infectious units, no defect was detected in the life cycle of mutant compared to wild-type virus. However, the resulting progeny virus particles were less infectious than the wild-type virions, and, when fibroblasts were infected with the same number of mutant or wild-type virus particles, a very early defect in the replication cycle was evident.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Biological reagents. Primary human foreskin fibroblasts were cultured in medium containing 10% newborn calf serum. Wild-type and mutant viruses were derived from an infectious bacterial artificial chromosome (BAC) clone, BADwt (41, 45), of the AD169 strain (35) of HCMV. The pUS24-deficient mutants BADinUS24 (insertion after sequence position 213931) and BADsubUS24 (substitution mutation between 214655 and 214733) were described previously by Yu et al. (46), where they were termed TN568 and DH65, respectively. BADrevUS24 is a revertant of BADinUS24 (46). Viruses were propagated, titered by plaque assay, and studied in fibroblasts.

To generate antibody to pUS24, glutathione S-transferase (GST) was fused to the N terminus of amino acids 303 to 482 of pUS24. The fusion protein was produced in Escherichia coli BL21 cells, purified, and used as an immunogen to generate murine hybridomas that were screened for interaction with the immunogen but not GST protein by Western blot assay. Additional monoclonal antibodies were used that recognized the following viral proteins: UL32-encoded pp150 (28); pUL44 (Virusys); UL55-encoded gB (gift from W. Britt, University of Alabama, Birmingham [7]); pUL69 (18), UL82-encoded pp71 (21); UL83-encoded pp65 (28), UL85-encoded major capsid protein, and UL86-encoded minor capsid protein (gifts from W. Gibson, Johns Hopkins University); UL99-coded pp28 (39); UL123-coded IE1 (A. Marchini, P. Robinson, and T. Shenk, unpublished); and pTRS1 (34). Rabbit polyclonal antibody to pp65 (gift from M. Schrader, University of Marburg) was employed for colocalization studies.

Purification and analysis of virions. To produce cell-free versus cell-associated virus preparations, fibroblasts were infected with BADwt, BADinUS24, or BADsubUS24 at a multiplicity of infection of 0.01 PFU/cell. After various time intervals, cell-free virus was isolated by collecting the medium and removing cells and cell debris by centrifugation, and cell-associated virus was prepared by lysing infected cells in fresh medium and removing debris by centrifugation.

Real-time PCR was used to quantify the amount of viral DNA present in aliquots of virus stocks. Virus particles were concentrated by centrifugation through a sorbitol cushion (20% D-sorbitol, 50 mM Tris-HCl [pH 7.2], 1 mM MgCl₂), and the infectivity present in concentrated samples was determined by plaque assay. Next, a portion of the concentrated virus containing 4 x 10⁴ PFU was treated with DNase I (2 U) at 37°C for 30 min to remove contaminating DNA outside of viral particles, and the reaction was stopped by adding DNase inactivation reagent (Ambion). Then the virus particles were incubated overnight at 37°C in lysis buffer (400 mM NaCl, 10 mM Tris [pH 8.0], 10 mM EDTA, 0.1 mg/ml proteinase K, 0.2% sodium dodecyl sulfate [SDS]), and virus DNA was extracted with phenol-chloroform and treated with 20 µg/ml RNase A for 1 h at 37°C. DNA was again extracted with phenol-chloroform, precipitated with ethanol, and resuspended in 40 µl 10 mM Tris (pH 7.8). Viral DNA corresponding to 5 x 10³ PFU was subjected to real-time PCR analysis using SYBR Green (Applied Biosystems) with a UL123-specific primer pair located at sequence positions 172101 (5'-CTGCAAACATCCTCCCATCA-3') and 172262 (5'-AATATACCCAGACGGAAGAGAAATTC-3') (43).

Virions were separated from noninfectious enveloped particles (NIEPs) and dense bodies (DBs) by centrifugation (4). Fibroblasts were infected at a multiplicity of infection of 0.01 PFU/cell and maintained in medium with 10% newborn calf serum for 10 to 12 days. The medium was collected and cleared of cells and cell debris by centrifugation, and virus particles were concentrated by centrifugation through a sorbitol cushion as described above. The pelleted virus particles were resuspended in TN (50 mM Tris [pH 7.4], 100 mM NaCl), layered onto a glycerol-tartrate gradient formed in TN, and centrifuged at 40,000 rpm for 15 min at 4°C. Virus particles were visualized with incandescent light and removed from the gradient. Particles were then pelleted by centrifugation, resuspended in a small volume of TN, and stored at –80°C. Aliquots (4 µg of total protein) were treated with trypsin (25 µg/ml) in 20 µl of TN supplemented with 1 mM CaCl₂ or treated with trypsin plus 1% Triton X-100 to remove the virion envelope and expose the tegument proteins to the protease (4). After incubation at 37°C for 1 h, trypsin digestions were terminated by adding 0.5 mM phenylmethylsulfonyl fluoride and 0.5 mg/ml soybean trypsin inhibitor (Sigma), and proteins were analyzed by Western blot assay.

Analysis of viral DNA, RNA, and protein within infected cells. Generally, total cell extracts were analyzed, but in some cases nuclear and cytoplasmic fractions were prepared and assayed. The fractionation was performed essentially as described by Kalesza and Shenk (23). Briefly, infected cells were suspended in RSB (10 mM NaCl, 3 mM MgCl₂, 10 mM Tris-HCl [pH 7.4]) and a 20x solution of detergent (10% sodium deoxycholate and 20% Tween 40 in RSB) was added to a final 1x concentration. Cells were agitated using a vortex mixer and incubated on ice for 5 min. Nuclei were pelleted by centrifugation at 1,000 x g for 3 min at 4°C. The supernatant and pellet were regarded as the cytoplasmic and nuclear fractions, respectively.

For analysis of viral DNA accumulation by slot blot assay (3), fibroblasts were harvested at various times after infection and lysed in buffer containing 25 mM NaCl, 10 mM Tris (pH 8.0), 25 mM EDTA, 0.5% SDS, and 1 mg/ml proteinase K. After incubation at 55°C for 3 h, DNA was extracted with phenol-chloroform, precipitated with ethanol, and resuspended in buffer containing 10 mM Tris (pH 8.0) and 25 mM EDTA. DNA was transferred onto a nylon membrane by using a slot blot apparatus (Schleicher and Schuell) and hybridized to a US22-specific, [³²P]dCTP-labeled DNA probe. After a wash, radioactivity retained on the membrane was quantified by using a phosphorimager.

For analysis of viral RNA accumulation by Northern blot assay, total RNA was prepared from fibroblasts at various times after infection using Trizol as recommended by the manufacturer (Invitrogen). Equivalent portions of RNA (5 µg) were glyoxylated, subjected to electrophoresis in a 1% agarose gel, and transferred to a nylon membrane. US23- or IE1-specific RNA was then detected with a strand-specific riboprobe to the US23 ORF (nucleotides 1 to 215) or UL123 exon 4 (23) using the SP6/T7 riboprobe combination system (Promega).

For analysis of viral proteins by Western blot assay, infected fibroblasts were dissolved in radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate). Protein was quantified by Bradford assay, and equal portions (50 µg) were subjected to electrophoresis in SDS-containing polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes and probed with antibodies to virus-encoded proteins. Goat anti-mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase was used as the secondary antibody.

To analyze infected-cell proteins by immunofluorescence, fibroblasts were grown on glass coverslips in six-well plates and harvested at various times after infection. All subsequent manipulations were carried out at room temperature. Cells were washed with phosphate-buffered saline (PBS), fixed for 15 min with 2% paraformaldehyde, washed again with PBS, and permeabilized for 15 min with PBS containing 0.1% Triton X-100. Cells were washed with PBS containing 0.2% Tween 20, incubated for 1 h with PBS blocking buffer containing 2% bovine serum albumin and 0.2% Tween 20, and then incubated in the same buffer for 1 h at room temperature with mouse monoclonal antibodies specific for virus-encoded proteins. After being washed with PBS containing 0.2% Tween 20, coverslips were incubated at room temperature for 30 min with a goat anti-mouse IgG-Alexa 546 conjugate (Molecular Probes). In some cases, the secondary antibody was supplemented with 1 µg/ml of DAPI (4',6'-diamidino-2-phenylindole; Molecular Probes) to stain DNA. For colocalization studies, mouse monoclonal antibody against US24 and rabbit polyclonal antibody against pp65 (gift from Martin Schrader, University of Marburg) were used in the primary-antibody incubation. Goat anti-mouse IgG-Alexa 546 and goat anti-rabbit IgG-Alexa 647 were used as secondary antibodies. Coverslips were mounted with Slow-Fade solution (Molecular Probes), and images were acquired by using a Zeiss LSM 510 confocal microscope.

Visualization of infected cells by electron microscopy. Fibroblasts were infected at a multiplicity of infection of 1 PFU/cell, and 96 h later cultures were washed with PBS and fixed with 3% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4) at room temperature for 1 h. Cells were collected, washed with 200 mM sodium cacodylate buffer, stained with 2% osmium tetroxide and 1% uranyl acetate, dehydrated, and then embedded in a blend of Embed 812, dodecyl succinic anhydride, and nadic methyl anhydride resins (Electron Microscopy Sciences). Thin sections were prepared and viewed at 80 kV with a Leo912a electron microscope.

Top Abstract Introduction Materials and Methods Results Discussion References

 
US24-deficient viruses generate reduced yields. To explore the function of pUS24, we studied two pUS24-deficient viruses that were produced by mutagenesis of an infectious BAC clone of HCMV strain AD169 (46). BADinUS24 contains a transposon with a marker cassette inserted into the US24 coding region, and BADsubUS24 contains a transposon with the same marker genes substituted for a portion of the US24 coding region (Fig. 1A).

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FIG. 1. pUS24-deficient mutants produce reduced yields compared to the wild-type virus. (A) Location of mutations. BADinUS24 contains a transposon insertion after base pair 1164 of the US24 ORF, and BADsubUS24 contains a transposon cassette with 79 base pairs (363 to 441) of the US24 ORF substituted. (B) Western blot analysis of pUS24. Fibroblasts were infected with wild-type (BADwt) and mutant viruses at a multiplicity of infection (MOI) of 1 PFU/cell and analyzed using a pUS24-specific monoclonal antibody at the indicated times. ß-Actin was monitored as a loading control. hpi, hours postinfection. (C) pUS24-deficient viruses exhibit a 20- to 30-fold growth defect compared to BADwt after infection of fibroblasts at an MOI that was relatively high (MOI = 1) or low (MOI = 0.01). At the indicated times, medium was collected from infected cultures and virus titers were determined by plaque assay. (D) Mutant viruses produce less cell-free and cell-associated virus. At the indicated times, cell-free and cell-associated virus was collected from cultures of infected fibroblasts, and the virus titer was determined by plaque assay. (E) BADrevUS24, a revertant derived from BADinUS24, exhibits wild-type growth. Infectivity in the medium was assayed by plaque assay at the indicated times. (F) US23 RNA accumulation is not disrupted in the two US24 mutants. US23-specific RNA was assayed by Northern blotting using a strand-specific riboprobe to the US23 ORF (nucleotides 1 to 215). 18S rRNA was monitored as a loading control.

We produced a monoclonal antibody to pUS24 and used it to monitor production of the protein after infection of fibroblasts (Fig. 1B). A 58-kDa protein, the predicted size for pUS24, was detected by Western blotting at 24 h after infection with BADwt, consistent with an earlier report that US24 is an early gene (10). The protein was not detected after infection with the mutants, confirming that their alterations prevented the accumulation of intact pUS24.

Both mutants produced 20- to 30-fold-less infectious extracellular virus (Fig. 1C), after infection at a relatively high (1 PFU/cell) or low (0.01 PFU/cell) input multiplicity, compared to their parent, BADwt, suggesting a functional role for pUS24 during replication in cultured fibroblasts. Relative levels of production of infectious intracellular versus extracellular virus also were compared after infection at a multiplicity of infection of 1 PFU/cell (Fig. 1D), and a similar defect in the production of infectious particles was evident for the mutant viruses in both compartments.

In our earlier study, revertant BADrevUS24 was made by allelic exchange in the BAC clone of BADinUS24, and it produced a virus stock with a wild-type titer (46). To confirm that the revertant replicates with wild-type kinetics, the growth of BADwt and that of BADrevUS24 were compared after infection of fibroblasts at a multiplicity of infection of 0.01 PFU/cell (Fig. 1E). The revertant grew indistinguishably from the wild-type virus, demonstrating that the growth defect was due to the mutation at the US24 locus. However, it remained possible that the mutation in US24 acted in cis to influence expression of an ORF to the right (US25 and US26) or left (US23) of the mutated ORF (Fig. 1A). A mutant with an insertion in the hypothetical US25 ORF has previously been shown to grow like wild-type virus, indicating that the mutation did not influence US26, where an insert generated a poorly growing virus (46). This demonstrates that a mutation to the left of US26 does not influence its expression sufficiently to generate a growth defect, and, as a consequence, the defect observed for the US24 mutations is not attributable to an effect on US26. US23 resides on the other side of US24, and a virus with a US23 mutation grows poorly (46). To monitor for an effect of the US24 mutations on US23 RNA accumulation, we performed a Northern blot assay using a US23-specific probe. A single RNA species was present at similar abundances in cells infected with wild-type compared to mutant viruses (Fig. 1F). Consequently, the BADinUS24 and BADsubUS24 growth defects almost certainly result from a failure to produce the US24 gene product.

We next searched for the point in the HCMV replication cycle within fibroblasts at which pUS24 is needed for the production of a normal yield. Initially, we examined the accumulation of representative viral proteins by Western blot assay. UL123-encoded IE1 (immediate-early), pUL44 (early), and UL99-encoded pp28 (late) accumulation was not decreased at any time assayed after infection with BADinUS24 compared to BADwt (Fig. 2A); thesame was true for UL55-encoded gB, pUL69, UL82-encoded pp71, and UL83-encoded pp65 (data not shown). The localization of IE1, pUL44, and pp28 (Fig. 2B), as well as gB and pp65 (data not shown), was examined by immunofluorescence at 72 h after infection, and there was no difference between mutant and wild-type virus infections. The accumulation of viral DNA was determined by slot blot analysis (Fig. 2C). There was no difference between wild-type and mutant viruses, consistent with the normal accumulation of pp28, a late protein whose expression depends on DNA replication, after infection with BADinUS24 (Fig. 2A).

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FIG. 2. pUS24-deficient virus appears normal when compared to wild-type virus after infection with equal numbers of infectious particles. (A) IE1, pUL44, and pp28 are expressed normally after infection with US24-deficient virus. Fibroblasts were mock infected or infected with BADwt or BADinUS24 at a multiplicity of infection of 1 PFU/cell, and the accumulation of viral proteins was assayed by Western blotting after various time intervals. (B) The cellular localizations of IE1, pUL44, and pp28 are normal after infection with US24-deficient virus. Fibroblasts were infected with BADwt or BADinUS24 at a multiplicity of infection of 0.3 PFU/cell, and viral proteins were assayed by immunofluorescence 72 h later (green, GFP; red, IE1, pUL44, or pp28). (C) Viral DNA accumulation is normal in the absence of pUS24. Fibroblasts were mock infected or infected at a multiplicity of infection of 1 PFU/cell, and virus DNA was assayed by slot blot at the indicated times.

Since viral proteins and DNA appeared to accumulate normally, we employed electron microscopy to evaluate the production and intracellular localization of virus particles. Fibroblasts were examined at 96 h after infection with BADwt (Fig. 3A) or BADinUS24 (Fig. 3B). Both wild-type- and mutant-virus-infected cells contained three types of capsids in the nuclei (A, empty; B, protein scaffold containing; C, DNA containing) and three types of enveloped particles in the cytoplasm (virions; NIEPs, and DBs). Further examination of multiple sections indicated that the different virus-specific particles were produced in approximately the same proportions after infection with the wild-type or mutant virus. For 357 virus particles in BADwt virus-infected cells, we observed 33.5% virions, 42% NIEPs, and 24.5% DBs; for 320 particles in BADinUS24 virus-infected cells, we observed 28.5% virions, 48% NIEPs, and 23.5% DBs. Thus, the production of virus particles that appear to be morphologically normal occurred in the absence of pUS24.

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FIG. 3. Electron microscopy shows that virus particles accumulate normally after infection with a pUS24-deficient virus. Fibroblasts were fixed and processed for imaging at 96 h after infection with BADwt or BADinUS24. Arrows identify C capsids in the nucleus and virions in the cytoplasm.

BADinUS24 virions are less infectious than BADwt virions. Since the infectious cycle of pUS24 mutants appeared normal, we tested the possibility that the particles produced by the mutant were less infectious than wild-type virions by determining the genome/PFU ratio. Wild-type and US24-deficient particles were partially purified by centrifugation through a sorbitol cushion, and the infectious titers of the preparations were determined. Then equal numbers of mutant and wild-type PFU were treated with DNase I to remove contaminating DNA outside of virus particles, and the amount of virus-specific DNA in the samples was quantified by using real-time PCR (Fig. 4A). The genome/PFU ratio for BADinUS24 was 10-fold higher than that for wild-type virus. Consistent with this observation, the input viral DNA of BADinUS24 was higher than that of BADwt in the slot blot analysis (Fig. 2C, 6 hours postinfection). When virions were separated from noninfectious HCMV particles by sedimentation in a glycerol-tartrate gradient, BADinUS24 virions exhibited an 15-fold-higher genome/PFU ratio than wild-type virions (data not shown).

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FIG. 4. Characterization of pUS24-deficient virions. (A, left) A pUS24-deficient mutant has a 10-fold-higher genome/PFU ratio than wild-type virus. The amounts of virus DNA present in 4 x 104 PFU of BADwt and BADinUS24 were determined by real-time PCR. Results are shown for undiluted and diluted (1:10) samples (in duplicate), and a sample with no added virus DNA was analyzed as a control. (A, right) Statistical summary of three independent experimental results. (B) pUS24 is a tegument protein. BADwt and BADinUS24 particles were purified, and equivalent amounts of protein from virions, NIEPs, and DBs were analyzed by Western blotting after no treatment (–), trysin digestion (T), or trypsin digestion after disruption of particles with Triton X-100 (TT). pUS24, pp28, and gB were assayed by Western blotting. (C) Colocalization of pUS24 and pp65 in the cytoplasm of infected fibroblasts. At 72 h after infection with BADwt, the localizations of virus proteins were assayed by immunofluorescence. pUS24 is represented by red, pp65 by green, and DAPI by blue; yellow marks colocalized pUS24 and pp65. (D) The levels of nine virion proteins are the same in BADinUS24 and BADwt virions. Equivalent amounts (normalized for the number of viral genomes) of gradient-purified virions were assayed by Western blotting using antibodies specific for the indicated virus-encoded proteins.

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FIG. 6. The virion proteins pp65 and pp71 move into the nucleus, but IE1 expression is reduced in BADinUS24-infected compared to BADwt-infected cells when cells receive equal numbers of particles. Fibroblasts were infected with aliquots of virus containing equal numbers of PFU or genomes. Cells were fixed and imaged by immunofluorescence at 2 h (pp65 and pp71) or 12 h postinfection (IE1).

Since the infectivity of the virions produced by the pUS24-deficient mutant was reduced, we examined the protein constituents of mutant and wild-type virions. Western blot analysis revealed that pUS24 is present in wild-type NIEPs, DBs, and virions; as expected, it was not present in mutant particles (Fig. 4B). To confirm that the pUS24 detected in the preparations was inside the virion envelope, particles were treated with trypsin prior to the Western blot assay, and pUS24 was not degraded (Fig. 4B, lane T). Control experiments demonstrated that the UL55-encoded gB envelope protein, which is partially exposed on the surface of the particles, was degraded by the trypsin treatment, whereas the internal UL99-encoded pp28 tegument protein was resistant. When particles were disrupted by treatment with Triton X-100, all assayed proteins became susceptible to trypsin treatment (Fig. 4B, lane TT). Since pUS24 is packaged into virions, we reasoned that it would accumulate within infected cells at the cytoplasmic assembly domain, as is the case for other virion proteins (36). At 72 h postinfection pUS24 and the UL83-encoded pp65 (8, 9, 38), a known virion tegument protein, were visualized by immunofluorescence, and they proved to colocalize (Fig. 4C). These experiments demonstrate that pUS24 is packaged within the envelopes of NIEPs, DBs, and virions, consistent with an earlier demonstration by mass spectroscopy that it is associated with purified virions (44).

To search for additional changes in the protein composition of BADinUS24 compared to BADwt particles, equivalent amounts of purified virions (normalized to equivalent genome copies) were subjected to Western blot assay. The nine HCMV virion proteins assayed were present in similar quantities in wild-type compared to BADinUS24 virions (Fig. 4D). Although BADinUS24 virions appeared to contain a reduced amount of pUL69 in the experiment displayed in Fig. 4, the difference was not reproduced in additional independent assays.

BADinUS24 exhibits a very early defect in comparison to BADwt after infection with the same number of virus particles. Our analysis of the genome-to-PFU ratio demonstrated that BADinUS24 virions are less infectious than BADwt particles. To identify the site in the HCMV infectious cycle where the infection initiated by mutant virions is compromised, we infected fibroblasts with virus preparations containing equal numbers of HCMV genomes and monitored the accumulation of representative virus proteins (Fig. 5A). The accumulation of IE1, pUL44, and pp28 was substantially delayed after infection with the mutant, demonstrating that the pUS24-deficient particles displayed a defect prior to the accumulation of an immediate-early protein. The production of IE1 mRNA also was delayed in mutant virus-infected cells (Fig. 5B), demonstrating that the defect occurred at the level of RNA accumulation.

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FIG. 5. The expression of immediate-early protein and RNA is delayed in cells infected with pUS24-deficient virus when cells receive equal numbers of particles. Viral protein (A) and mRNA (B) expression was delayed after infection with BADinUS24 compared to BADwt. Cells received particles containing the same number of mutant or wild-type genomes. At the indicated times IE1, pUL44, and pp28 were assayed by Western blotting and IE1 mRNA was assayed by Northern blotting. ß-Actin protein and 18S rRNA were monitored as loading controls. hpi, hours postinfection.

We next investigated the possibility that BADinUS24 virions enter fibroblasts less efficiently than BADwt particles. Entry was monitored by assaying the nuclear accumulation of the pp65 tegument protein delivered into cells by infecting virions. When the same number of wild-type or mutant particles was used to infect fibroblasts, equivalent numbers of nuclei contained pp65 at 2 h postinfection (Fig. 6, equal input particles), arguing that entry of the BADinUS24 tegument plus capsid and dissociation of pp65 from the capsid are normal. Similar results were obtained for a second tegument protein, pp71. When the mutant inoculum was increased so the fibroblasts received same number of mutant and wild-type PFU, pp65 was present in a significantly higher number of nuclei after infection with BADinUS24 compared to BADwt (Fig. 6, equal input PFU). IE1 also was monitored in this experiment. As expected, the immediate-early protein accumulated in the same number of nuclei after infection with mutant or wild-type viruses when equivalent numbers of PFU were used, and it accumulated in fewer nuclei when equivalent numbers of particles were used.

In addition to tegument proteins, we assayed the movement of viral genomes into the nucleus. Nuclear and cytoplasmic fractions were prepared at 2 h after infection, and their successful separation was demonstrated by monitoring the location of input cytoplasmic UL99-encoded pp28 and nuclear UL83-encoded pp65 (Fig. 7A). The location of the viral genome in these two fractions was then assayed by real-time PCR using primers specific to the UL123 ORF. The amount of input DNA was normalized by monitoring the cellular -actin gene. There was no difference in the amounts of viral DNA present in the nuclei of cells infected with BADinUS24 compared to BADwt (Fig. 7B).

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FIG. 7. Viral genomes move to the nucleus in BADinUS24-infected cells as efficiently as in BADwt-infected cells. (A) Separation of nuclear and cytoplasmic fractions. At 2 h postinfection, infected fibroblasts were collected and fractionated. Equal amounts of protein from the total cell lysate, the nuclear fraction, and cytoplasmic fraction were assayed for pp65 and pp28 by Western blotting. (B) Similar amounts of viral DNA accumulate in the nucleus after infection with equal numbers of mutant and wild-type particles. Viral DNA was quantified by real-time PCR analysis using a primer pair specific to the UL123 ORF.

BADinUS24 virions exhibit a defect after the viral genome and at least two virion tegument proteins reach the nucleus and before the accumulation of IE1 protein.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The HCMV AD169 genome comprises two blocks of single-copy ORFs: the unique long (UL) and unique short (US) domains. US24 is one of only three ORFs in the US region that significantly enhance the replication of AD169 in fibroblasts (46). Our present study demonstrates that two different mutant viruses (Fig. 1A) unable to express pUS24 (Fig. 1B) generate 20- to 30-fold less infectious progeny than their wild-type AD169 parent (Fig. 1C). We also constructed a third mutant, in which the entire US24 ORF is deleted, and it grew like the two mutants characterized in this report (data not shown). Dunn et al. (14) reported that a US24-deficient mutant of the Towne strain of HCMV grew as well as wild-type virus in fibroblasts. Possibly, the difference in virus strains accounts for the different results.

Even though the mutant viruses produced a reduced infectious yield, no difference was evident when different stages of the HCMV replication cycle were assayed after infection of fibroblasts with equal amounts of mutant or wild-type virus (Fig. 2 and 3). Rather, mutant virions were about 10-fold less infectious than wild-type particles (Fig. 4A). Since pUS24 is a virion protein (Fig. 4B) (44), it is not surprising that the lack of pUS24 could influence the infectivity of virions. Mutant viruses lacking other tegument proteins, e.g., pUL47 (5) and pUL35 (37), also make virions with abnormally high particle-to-PFU ratios.

The reduced infectivity of BADinUS24 virions could be a direct consequence of the pUS24 deficiency, i.e., the protein might function directly to facilitate infection after it is delivered to cells in the virion. Alternatively, pUS24 could influence the appropriate assembly of virions. We did not observe significant quantitative differences in the virion protein constituents that were assayed (Fig. 4D), but it is possible that other proteins are altered. Further, virion proteins might be assembled incorrectly in the absence of pUS24, even though they are present in normal proportions.

We tested the possibility that a change in virion composition or organization might cause the mutant particles to become less stable. The infectivity of BADwt and BADinUS24 was assayed after virus stocks were maintained at 37°C for various periods of time (data not shown). No difference between the mutant and wild type in terms of decay of virion infectivity was evident.

To explore the basis for the reduced infectivity of US24-deficient virus particles, we monitored events in the virus replication cycle after infection of fibroblasts with equal numbers of mutant and wild-type particles. The tegument proteins that were assayed (pp65 and pp71; Fig. 6) and the viral genome (Fig. 7) were delivered normally into the nucleus after infection with BADinUS24. However, viral gene expression was delayed. IE1 mRNA (Fig. 5B) and protein (Fig. 5A) accumulated after a substantial delay in mutant-virus- compared to wild-type-virus-infected cells. By immunofluorescence, the number of IE1-positive cells was decreased (67%) at 12 h after infection with the mutant versus wild-type virus (Fig. 6). Thus, US24-deficient virions exhibit a delay in the accumulation of immediate-early gene expression, even though the viral DNA and at least some tegument proteins have reached their appropriate intracellular locations.

. . . . . .

 
We thank W. Britt (University of Alabama, Birmingham), W. Gibson (Johns Hopkins University School of Medicine), and M. Schrader (University of Marburg) for kind gifts of antibodies.

 
* Corresponding author. Mailing address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014. Phone: (609) 258-5992. Fax: (609) 258-1704. E-mail: tshenk{at}princeton.edu.

Present address: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, September 2006, p. 8371-8378, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00399-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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