Proteins Specified by Herpes Simplex Virus VI. Viral Proteins in the Plasma Membrane

Previous papers in the series have shown that the surface membranes of herpesvirus-infected cells acquire new immunological specificities and that purified infected cell membrane preparations, characterized by their physical properties rather than topology in the cell, contain new glycoproteins genetically determined by the virus. In this study, we prepared purified plasma membrane identified by its 5' nucleotidase, fucose, and reduced nicotinamide adenine dinucleotide-diaphorase content. Analysis of the membrane proteins and glycoproteins by electrophoresis in acrylamide gels indicated the following. (i) Purified plasma membranes from infected cells contained two sets of proteins, i.e., host proteins were present both before and after infection and viral proteins were present only after infection. (ii) After infection, no appreciable selective or nonselective loss of host proteins from membranes was demonstrable. However, no new host proteins were made. (iii) Electropherograms of plasma membrane proteins from infected cells indicated the presence of at least 12 virus-specific proteins ranging in molecular weight from 25 x 10(3) to 126 x 10(3) daltons. Of these, at least nine were glycosylated. Proteins and glycoproteins with similar electrophoretic mobilities but in somewhat different ratios were also present in preparations of highly purified virions.     

Temperature-Sensitive Simian Virus 40 Mutant Defective in a Late Function

A temperature-sensitive simian virus 40 (SV40) mutant, tsTNG-1, has been isolated from nitrosoguanidine-treated and SV40-infected African green monkey kidney (CV-1) cultures. Replication of virus at the nonpermissive temperature (38.7 C) was 3,000-fold less than at the permissive temperature (33.5 C). Plaque formation by SV40tsTNG-1 deoxyribonucleic acid (DNA) on CV-1 monolayers occurred normally at 33.5 C but was grossly inhibited at 38.7 C. The time at which virus replication was blocked at 38.7 C was determined by temperature-shift experiments. In shift-up experiments, cultures infected for various times at 33.5 C were shifted to 38.7 C. In shift-down experiments, cultures infected for various times at 38.7 C were shifted to 33.5 C. All cultures were harvested at 96 hr postinfection (PI). No virus growth occurred when the shift-up occurred before 40 hr PI. Maximum virus yields were obtained at 96 hr PI when the shift-down occurred at 66 hr, but only about 15% of the maximum yield was obtained when the shift-down occurred at 76 hr PI. These results indicate that SV40tsTNG-1 contains a conditional lethal mutation in a late viral gene function. Mutant SV40tsTNG-1 synthesized T antigen, viral capsid antigens, and viral DNA, and induced thymidine kinase activity at either 33.5 or 38.7 C. The properties of the SV40 DNA synthesized in mutant-infected CV-1 cells at 33.5 or 38.7 C were very similar to those of SV40 DNA made in parental virus-infected cells, as determined by nitrocellulose column chromatography, cesium-chloride-ethidium bromide equilibrium centrifugation, and by velocity centrifugation in neutral sucrose gradients. Mutant SV40tsTNG-1 enhanced cellular DNA synthesis in primary cultures of mouse kidney cells at 33.5 and 38.7 C and also transformed mouse kidney cultures at 36.5 C. SV40tsTNG-1 was recovered from clonal lines of transformed cells after fusion with susceptible CV-1 cells and incubation of heterokaryons at 33.5 C, but not at 38.7 C.     

Packaging-Competent Capsids of a Herpes Simplex Virus Temperature-Sensitive Mutant Have Properties Similar to Those of In Vitro-Assembled Procapsids

Newcomb and coworkers (W. W. Newcomb, F. L. Homa, D. R. Thomsen, F. P. Booy, B. L. Trus, A. C. Steven, J. V. Spencer, and J. C. Brown, J. Mol. Biol. 263:432-446, 1996; W. W. Newcomb, F. L. Homa, D. R. Thomsen, Z. Ye, and J. C. Brown, J. Virol. 68:6059-6063, 1994) have recently described an in vitro herpes simplex virus (HSV) capsid assembly product which, because of certain parallels between its properties and those of bacteriophage proheads, they have designated the procapsid. As in their bacteriophage counterparts, there are marked differences between the structures of the two types of particle, and conversion from the procapsid to the capsid form requires extensive reconfiguration of the subunits. This reconfiguration occurs spontaneously upon extended in vitro incubation. One of the distinctive features of the HSV procapsids is that, unlike mature capsids, they are unstable and disassemble upon storage at 2 degrees C. Using a mutant of HSV type 1 (ts1201), which has a lesion in the protease responsible for maturational cleavage of the scaffolding protein, we have demonstrated that capsids present within cells infected at nonpermissive temperatures are also cryosensitive and disappear if the cells are incubated at 0 degrees C. This suggests that ts1201 capsids may resemble procapsids in structure. However, ts1201 capsids remain cryosensitive following extended incubation at an elevated temperature and, therefore, do not appear to undergo the spontaneous reconfiguration seen with in vitro-assembled procapsids. The lesion in ts1201 is reversible, and capsids formed at the nonpermissive temperature can undergo maturational cleavage and go on to form infectious virions following downshift to permissive temperatures. The sensitivity of ts1201 capsids to low temperatures is closely correlated with the cleavage status of the scaffolding protein, suggesting that proteolysis may act to trigger their conversion to the stable form. The experiments described here provide the firmest evidence yet that the procapsid has a biologically relevant role in the virus life cycle.     

Expression of the Viral Thymidine Kinase Gene in Herpes Simplex Virus-Transformed L Cells

In these studies, the expression of thymidine kinase (TK) in normal and herpes simplex virus (HSV)-transformed L cells has been compared. In asynchronously dividing cultures of L cells, the TK activity rose and declined rapidly and coordinately with DNA synthesis. When net cell increase stopped, TK activity was at a minimum. In contrast, TK activity of HSV-transformed cells remained at a minimum during rapid DNA synthesis and gradually increased as the rate of DNA synthesis decreased. When net cell increase stopped, TK activity was at a maximum. In synchronous cultures of L cells, TK activity rose and fell coordinately with the rate of DNA synthesis. In synchronous cultures of HSV-transformed cells, no increase in TK activity was observed during the period of rapid DNA synthesis, i.e., the S phase. These findings indicated that the viral TK gene in HSV-transformed cells was not placed under the control of the cellular mechanisms which normally modulate the host cell TK gene. Lytic infection of HSV-transformed cells with a TK(-) mutant of HSV-1 induced a four-to fivefold increase in viral TK. The TK of HSV-1 was induced in the HSV-1-transformed cells and HSV-2 in the HSV-2-transformed cells by this TK(-) mutant. The same infection of normal L cells decreased the cellular TK activity by 80%. This stimulation, rather than inhibition, suggest that the viral gene in HSV-transformed cells retain some of its original viral characteristics.     

Identification and Characterization of a Herpes Simplex Virus Gene Product Required for Encapsidation of Virus DNA

A mutant of herpes simplex virus type 1, 17tsVP1201, has a temperature-sensitive processing defect in a late virus polypeptide. Immunoprecipitation studies with monoclonal antibodies showed that the aberrant polypeptide in mutant virus-infected cells was the nucleocapsid polypeptide known as p40. Since a revertant, TS(+) for growth, processed the polypeptide normally under conditions restrictive for the mutant, the processing event must be essential for virus replication. Electron microscopic analysis of mutant virus-infected cells grown at the nonpermissive temperature revealed that the nuclei contained large aggregations of empty nucleocapsids possessing some internal structure. Therefore, although the mutant synthesized virus DNA at the nonpermissive temperature, the DNA was not packaged into nucleocapsids. When mutant virus-infected cells were shifted from 39 to 31 degrees C in the presence of cycloheximide, the polypeptide p40 was processed to lower-molecular-weight forms, and full nucleocapsids were detected in the cell nuclei. The aberrant polypeptide of the mutant, however, was not processed in cells mixedly infected with 17tsVP1201 and a revertant at the nonpermissive temperature, suggesting that the defect of the mutant was in the gene encoding p40 rather than in a gene of a processing enzyme.     

Molecular Genetics of Herpes Simplex Virus II. Mapping of the Major Viral Glycoproteins and of the Genetic Loci Specifying the Social Behavior of Infected Cells

We have mapped the location in herpes simplex virus (HSV) DNA of (i) three mutations at different loci (syn loci) which alter the social behavior of infected cells from clumping of rounded cells to polykaryocytosis, (ii) a mutation which determines the accumulation of one major glycoprotein [VP8.0(C(2))], and (iii) the sequences encoding four major virus glycoproteins [VP8.0(C(2)), VP7(B(2)), VP8.5(A), and VP19E(D(2))]. The experimental design and results were as follows. (i) Analysis of HSV-1 x HSV-2 recombinants showed that the sequences encoding the VP19E(D(2)) glycoprotein map in the S component, whereas the sequences encoding the other three major glycoproteins are in two locations in the L component of HSV DNA. The templates specifying the HSV-1 and HSV-2 glycoprotein VP8.0(C(2)) appear not to be colinear; we isolated recombinants specifying glycoproteins comigrating in sodium dodecyl sulfate-polyacrylamide gels with VP8.0(C(2)) of both HSV-1 and HSV-2. (ii) Marker rescue of a ts mutant defective in accumulation of glycoprotein VP7(B(2)) showed that the mutation maps within a region containing the sequences encoding that glycoprotein. (iii) Marker transfer experiments involving transfection of rabbit skin cells with donor HSV-1(F) DNA and fragments from several donor strains causing fusion of Vero or both Vero and HEp-2 cells revealed the existence of three syn loci specifying the social behavior of cells and one locus (Cr) determining the accumulation of glycoprotein VP8.0(C(2)). The Cr locus maps to the right of the template specifying VP8.0(C(2)) glycoprotein. Loci syn 1 and syn 2 map at or near the Cr locus but can be segregated from it. Locus syn 3 maps at or near the template specifying glycoproteins VP7(B(2)) and VP8.5(A). The expression of mutations in the syn 1 and syn 3 loci appear to be cell type dependent, in that recombinants with these mutations fuse Vero cells but not HEp-2 cells. Recipients of the syn 2 locus or of both syn 2 and syn 1 loci fuse both Vero and HEp-2 cells.     

Early Events in Herpes Simplex Virus Infection: a Radioautographic Study

The early events in herpes simplex virus infection were studied by means of radio-autography. The virus was rapidly taken up by the host cells and uncoated. Viral deoxyribonucleic acid (DNA) reached the nuclear sites of replication in 15 to 30 min after infection. The viral DNA occasionally associated with chromosomes or condensed chromatin but was more frequently found to be randomly distributed. Viral progeny appeared 3 hr after infection. These particles did not show any particular spatial relationship to the parental DNA. The morphological latent period lasted 2.5 hr.     

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus VI. Polyadenylic Acid Sequences in Viral Messenger Ribonucleic Acid

Evidence is presented by use of radiolabeling and pancreatic and T1 ribonuclease digestion that some of the ribonucleic acid specified by herpes simplex virus contains polyadenylic acid sequences. The polyadenylic sequences are not transcribed from viral DNA.     

A Herpes Simplex Virus Scaffold Peptide That Binds the Portal Vertex Inhibits Early Steps in Viral Replication

Previous experiments identified a 12-amino-acid (aa) peptide that was sufficient to interact with the herpes simplex virus 1 (HSV-1) portal protein and was necessary to incorporate the portal into capsids. In the present study, cells were treated at various times postinfection with peptides consisting of a portion of the Drosophila antennapedia protein, previously shown to enter cells efficiently, fused to either wild-type HSV-1 scaffold peptide (YPYYPGEARGAP) or a control peptide that contained changes at positions 4 and 5. These 4-tyrosine and 5-proline residues are highly conserved in herpesvirus scaffold proteins and were previously shown to be critical for the portal interaction. Treatment early in infection with subtoxic levels of wild-type peptide reduced viral infectivity by over 1,000-fold, while the mutant peptide had little effect on viral yields. In cells infected for 3 h in the presence of wild-type peptide, capsids were observed to transit to the nuclear rim normally, as viewed by fluorescence microscopy. However, observation by electron microscopy in thin sections revealed an aberrant and significant increase of DNA-containing capsids compared to infected cells treated with the mutant peptide. Early treatment with peptide also prevented formation of viral DNA replication compartments. These data suggest that the antiviral peptide stabilizes capsids early in infection, causing retention of DNA within them, and that this activity correlates with peptide binding to the portal protein. The data are consistent with the hypothesis that the portal vertex is the conduit through which DNA is ejected to initiate infection.     

Complementation of a Herpes Simplex Virus ICP0 Null Mutant by Varicella-Zoster Virus ORF61p

The herpes simplex virus (HSV) ICP0 protein acts to overcome intrinsic cellular defenses that repress viral α gene expression. In that vein, viruses that have mutations in ICP0''s RING finger or are deleted for the gene are sensitive to interferon, as they fail to direct degradation of promyelocytic leukemia protein (PML), a component of host nuclear domain 10s. While varicella-zoster virus is also insensitive to interferon, ORF61p, its ICP0 ortholog, failed to degrade PML. A recombinant virus with each coding region of the gene for ICP0 replaced with sequences encoding ORF61p was constructed. This virus was compared to an ICP0 deletion mutant and wild-type HSV. The recombinant degraded only Sp100 and not PML and grew to higher titers than its ICP0 null parental virus, but it was sensitive to interferon, like the virus from which it was derived. This analysis permitted us to compare the activities of ICP0 and ORF61p in identical backgrounds and revealed distinct biologic roles for these proteins.Alphaherpesviruses encode orthologs of the herpes simplex virus (HSV) α gene product ICP0. ICP0 is a nuclear phosphoprotein that behaves as a promiscuous activator of viral and cellular genes (7, 11, 28, 29). ICP0 also functions as an E3 ubiquitin ligase to target several host proteins for proteasomal degradation (4, 10, 11, 16, 26). Through this activity, ICP0 promotes degradation of components of nuclear domain 10 (ND10) bodies, including the promyelocytic leukemia protein (PML) and Sp100. These proteins are implicated in silencing of herpesvirus genomes (9, 10, 22, 34). Therefore, ICP0-mediated degradation of ND10 components may disrupt silencing of HSV genes to enable efficient gene expression. This hypothesis provides a plausible mechanistic explanation of how ICP0 induces gene activation.Introduction of DNA encoding the ICP0 orthologs from HSV, bovine herpesvirus, equine herpesvirus, and varicella-zoster virus (VZV) can also affect nuclear structures and proteins (27). In addition, and more specific to this report, ORF61p, the VZV ortholog, activates viral promoters and enhances infectivity of viral DNA like ICP0, the prototype for this gene family (24, 25). However, we have previously demonstrated two key biological differences between the HSV and VZV orthologs. We first showed that unlike ICP0, ORF61p is unable to complement depletion of BAG3, a host cochaperone protein. As a result, VZV is affected by silencing of BAG3 (15), whereas growth of HSV is altered only when ICP0 is not expressed (17). Furthermore, we have shown that while both proteins target components of ND10s, expression of ICP0 results in degradation of both PML and Sp100, whereas ORF61p specifically reduces Sp100 levels (16). These findings suggest that these proteins have evolved separately to provide different functions for virus replication.Virus mutants lacking the ICP0 gene have an increased particle-to-PFU ratio, a substantially lower yield, and decreased levels of α gene expression, in a multiplicity-of-infection (MOI)- and cell-type-dependent manner (2, 4, 8, 33). These mutants are also defective at degrading ND10 components (23). Depletion of PML and Sp100 accelerates virus gene expression and increases plaquing efficiency of HSV ICP0-defective viruses but has no effect on wild-type virus, suggesting that PML and Sp100 are components of an intrinsic anti-HSV defense mechanism that is counteracted by ICP0''s E3 ligase activity (9, 10). Interestingly, ICP0 null viruses are also hypersensitive to interferon (IFN) (26), a property that was suggested to be mediated via PML (3).To directly compare the activities of the two orthologs, we constructed an HSV mutant virus that expresses ORF61p in place of ICP0. The resulting chimeric virus only partially rescues the ICP0 null phenotype. Our studies emphasize the biological differences between ICP0 and ORF61p and shed light on the requirements for PML and Sp100 during infection.     

Pocket Protein p130/Rb2 Is Required for Efficient Herpes Simplex Virus Type 1 Gene Expression and Viral Replication

We have reported previously that herpes simplex virus type 1 (HSV-1) infection disrupts normal progression of the mammalian cell cycle, causing cells to enter a G(1)-like state. Infected cells were characterized by a decline in cyclin-dependent kinase 2 (CDK2) activities, loss of hyperphosphorylated retinoblastoma protein (pRb), accumulation of E2F-pocket protein complexes, and failure to initiate cellular DNA replication. In the present study, we investigated the role of the pocket proteins pRb, p107, and p130 in HSV-1-dependent cell cycle inhibition and cyclin kinase regulation by infecting murine 3T3 cells derived from wild-type (WT) mouse embryos or embryos with deletions of pRb (pRb(-/-)), p107 (p107(-/-)), p130 (p130(-/-)), or both p130 and p107 (p130(-/-)/p107(-/-)). With respect to CDK2 inhibition, viral protein accumulation, viral DNA replication, and progeny virus yield, WT, pRb(-/-), and p107(-/-) cells were essentially identical. In contrast, after infection of p130(-/-) cells, we observed no inhibition of CDK2 activity, a 5- to 6-h delay in accumulation of viral proteins, an impaired ability to form viral DNA replication compartments, and reduced viral DNA synthesis. As a result, progeny virus yield was reduced 2 logs compared to that in WT cells. Notably, p130(-/-)/p107(-/-) double-knockout cells had a virus replication phenotype intermediate between those of the p107(-/-) and p130(-/-) cells. We conclude from these studies that p130 is a key factor in regulating aspects of cell cycle progression, as well as the timely expression of viral genes and replication of viral DNA.     

Characterization of the Growth of Herpes Simplex Virus in Human Lymphoid Cells

Herpes simplex virus was grown in a 6-liter suspended culture of an atypical permanent human lymphoid cell line, Roswell Park Memorial Institute no. 8226. The kinetics of virus replication were determined by counting viruses by electron microscopy, plaque formation, and tissue culture infectivity. Deoxyribonucleic acid-dependent deoxyribonucleic acid polymerase activity was determined during the course of infection. Electron microscopy studies substantiated the kinetics of the virus infection in lymphoid cells.     

Isolation and Characterization of the Herpes Simplex Virus 1 Terminase Complex

During herpes simplex virus 1 (HSV-1) infection, empty procapsids are assembled and subsequently filled with the viral genome by means of a protein complex called the terminase, which is comprised of the HSV-1 UL15, UL28, and UL33 proteins. Biochemical studies of the terminase proteins have been hampered by the inability to purify the intact terminase complex. In this study, terminase complexes were isolated by tandem-affinity purification (TAP) using recombinant viruses expressing either a full-length NTAP-UL28 fusion protein (vFH476) or a C-terminally truncated NTAP-UL28 fusion protein (vFH499). TAP of the UL28 protein from vFH476-infected cells, followed by silver staining, Western blotting, and mass spectrometry, identified the UL15, UL28, and UL33 subunits, while TAP of vFH499-infected cells confirmed previous findings that the C terminus of UL28 is required for UL28 interaction with UL33 and UL15. Analysis of the oligomeric state of the purified complexes by sucrose density gradient ultracentrifugation revealed that the three proteins formed a complex with a molecular mass that is consistent with the formation of a UL15-UL28-UL33 heterotrimer. In order to assess the importance of conserved regions of the UL15 and UL28 proteins, recombinant NTAP-UL28 viruses with mutations of the putative UL28 metal-binding domain or within the UL15 nuclease domain were generated. TAP of UL28 complexes from cells infected with each domain mutant demonstrated that the conserved cysteine residues of the putative UL28 metal-binding domain and conserved amino acids within the UL15 nuclease domain are required for the cleavage and packaging functions of the viral terminase, but not for terminase complex assembly.