UL34 Proteins Research Articles

Temperature-Sensitive Mutations in the Putative Herpes Simplex Virus Type 1 Terminase Subunits pU L 15 and pU L 33 Preclude Viral DNA Cleavage/Packaging and Interaction with pU L 28 at the Nonpermissive Temperature

Terminases comprise essential components of molecular motors required to package viral DNA into capsids in a variety of DNA virus systems. Previous studies indicated that the herpes simplex virus type 1 U(L)15 protein (pU(L)15) interacts with the pU(L)28 moiety of a pU(L)28-pU(L)33 complex to form the likely viral terminase. In the current study, a novel temperature-sensitive mutant virus was shown to contain a mutation in U(L)33 codon 61 predicted to change threonine to proline. At the nonpermissive temperature, this virus, designated ts8-22, replicated viral DNA and produced capsids that became enveloped at the inner nuclear membrane but failed to form plaques or to cleave or package viral DNA. Incubation at the nonpermissive temperature also precluded coimmunoprecipitation of U(L)33 protein with its normal interaction partners encoded by U(L)28 and U(L)15 in ts8-22-infected cells and with pU(L)28 in transient-expression assays. Moreover, a temperature-sensitive mutation in U(L)15 precluded coimmunoprecipitation of pU(L)15 with the U(L)28 and U(L)33 proteins at the nonpermissive temperature. We conclude that interactions between putative terminase components are tightly linked to successful viral DNA cleavage and packaging.

U S 3 of Herpes Simplex Virus Type 1 Encodes a Promiscuous Protein Kinase That Phosphorylates and Alters Localization of Lamin A/C in Infected Cells

The herpes simplex virus type 1 (HSV-1) US3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins UL31 and UL34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins UL34 and UL31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of US3 kinase activity. To determine how US3 alters lamin A/C distribution, US3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative US3 kinase consensus site in the lamin A/C sequence. US3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires US3 kinase activity. These data suggest that US3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.

A conserved interaction between the Heterogeneous Nuclear Ribonucleoprotein K and the HSV-1 proteins UL34 and UL31 is required for capsid egress

A conserved interaction between the Heterogeneous Nuclear Ribonucleoprotein K and the HSV-1 proteins UL34 and UL31 is required for capsid egress

RNA Binding by the Herpes Simplex Virus Type 1 Nucleocytoplasmic Shuttling Protein UL47 Is Mediated by an N-Terminal Arginine-Rich Domain That Also Functions as Its Nuclear Localization Signal

The function of the alphaherpesvirus UL47 tegument protein has not yet been defined. Nonetheless, previous studies with transfected cells have shown that both the herpes simplex virus type 1 homologue (hUL47, or VP13/14) and the bovine herpesvirus type 1 (BHV-1) homologue (bUL47, or VP8) have the capacity to shuttle between the nucleus and the cytoplasm. Furthermore, hUL47 packaged into the virion has also been shown to bind several individual virus-specific RNA transcripts. Here, we extend these observations and show that hUL47 binds a wide range of RNA species in vitro. It has a high affinity for polyadenylated transcripts but has no apparent selectivity for virus-encoded RNA over cellular RNA. We also show that the virion population of bUL47 binds RNA in vitro. However, while purified recombinant hUL47 retains its RNA binding activity, recombinant bUL47 does not, suggesting that the BHV-1 homologue may require virus-induced modification for its activity. We identify the minimal RNA binding domain in hUL47 as a 26-residue N-terminal peptide containing an arginine-rich motif that is essential but not sufficient for optimal RNA binding, and we demonstrate that this RNA binding domain incorporates the hUL47 minimal nuclear localization signal. In addition, we show that soon after hUL47 is expressed during infection, it colocalizes in the infected cell nucleus with ICP4, the major virus transcriptional activator. Using RNA immunoprecipitations, we demonstrate that hUL47 is also bound in vivo to at least one viral transcript, the ICP0 mRNA. Taken together, these results suggest that hUL47 may play a role in RNA biogenesis in the infected cell.

Linker Insertion Mutations in the Herpes Simplex Virus Type 1 UL28 Gene: Effects on UL28 Interaction with UL15 and UL33 and Identification of a Second-Site Mutation in the UL15 Gene That Suppresses a Lethal UL28 Mutation

The UL28 protein of herpes simplex virus type 1 (HSV-1) is one of seven viral proteins required for the cleavage and packaging of viral DNA. Previous results indicated that UL28 interacts with UL15 and UL33 to form a protein complex (terminase) that is presumed to cleave concatemeric DNA into genome lengths. In order to define the functional domains of UL28 that are important for DNA cleavage/packaging, we constructed a series of HSV-1 mutants with linker insertion and nonsense mutations in UL28. Insertions that blocked DNA cleavage and packaging were found to be located in two regions of UL28: the first between amino acids 200 to 400 and the second between amino acids 600 to 740. Insertions located in the N terminus or in a region located between amino acids 400 and 600 did not affect virus replication. Insertions in the carboxyl terminus of the UL28 protein were found to interfere with the interaction of UL28 with UL33. In contrast, all of the UL28 insertion mutants were found to interact with UL15 but the interaction was reduced with mutants that failed to react with UL33. Together, these observations were consistent with previous conclusions that UL15 and UL33 interact directly with UL28 but interact only indirectly with each other. Revertant viruses that formed plaques on Vero cells were detected for one of the lethal UL28 insertion mutants. DNA sequence analysis, in combination with genetic complementation assays, demonstrated that a second-site mutation in the UL15 gene restored the ability of the revertant to cleave and package viral DNA. The isolation of an intergenic suppressor mutant provides direct genetic evidence of an association between the UL28 and UL15 proteins and demonstrates that this association is essential for DNA cleavage and packaging.

Identification of the functional domains of the essential human cytomegalovirus UL34 proteins

The human cytomegalovirus UL34 gene represses expression of the US3 immune evasion gene and is essential for viral replication in cell culture. Two highly related proteins, an early and a late protein, are synthesized at different times during infection from the UL34 gene. The late protein differs from the early protein only by the omission of 21 amino terminal amino acids. Both UL34 proteins repress expression of the US3 promoter and bind specifically to a DNA element in the US3 gene. We have localized the DNA-binding domain of the UL34 open reading frame to amino acids 22 to 243. Surprisingly, this same region of the UL34 open reading frame was also sufficient for transcriptional repression of US3 expression. The UL34 gene is unusual in encoding proteins that have extensively overlapping DNA-binding and transcriptional regulatory domains.

The Putative Terminase Subunit of Herpes Simplex Virus 1 Encoded by U L 28 Is Necessary and Sufficient To Mediate Interaction between pU L 15 and pU L 33

Viral terminases play essential roles as components of molecular motors that package viral DNA into capsids. Previous results indicated that the putative terminase subunits of herpes simplex virus 1 (HSV-1) encoded by U(L)15 and U(L)28 (designated pU(L)15 and pU(L)28, respectively) coimmunoprecipitate with the U(L)33 protein from lysates of infected cells. All three proteins are among six required for HSV-1 DNA packaging but dispensable for assembly of immature capsids. The current results show that in both infected- and uninfected-cell lysates, pU(L)28 coimmunoprecipitates with either pU(L)33 or pU(L)15, whereas pU(L)15 and pU(L)33 do not coimmunoprecipitate unless pU(L)28 is present. The U(L)28 protein was sufficient to stabilize pU(L)33 from proteasomal degradation in an engineered cell line and was necessary to stabilize pU(L)33 in infected cells, whereas pU(L)15 had no such effects. The presence of pU(L)33 was dispensable for the pU(L)15/pU(L)28 interaction in lysates of both infected and uninfected cells but augmented the tendency for pU(L)15 and pU(L)28 to coimmunoprecipitate. These data suggest that pU(L)28 and pU(L)33 interact directly and that pU(L)15 interacts directly with pU(L)28 but only indirectly with pU(L)33. It is logical to propose that the indirect interaction of pU(L)15 and pU(L)33 is mediated through the interaction of both proteins with pU(L)28. The data also suggest that one function of pU(L)33 is to optimize the pU(L)15/pU(L)28 interaction.

Interactions among Four Proteins Encoded by the Human Cytomegalovirus UL112-113 Region Regulate Their Intranuclear Targeting and the Recruitment of UL44 to Prereplication Foci

Four phosphoproteins, of 34, 43, 50, and 84 kDa, with common amino termini are synthesized via alternative splicing from the UL112-113 region of the human cytomegalovirus genome. Although genetic studies provided evidence that both the UL112 and UL113 loci in the viral genome are required for efficient viral replication, whether the four proteins play specific roles or cooperate in replication is not understood. Here we present evidence, using in vitro and in vivo coimmunoprecipitation assays, that the four UL112-113 proteins both self-interact and interact with each other. A mapping study of the 84-kDa protein showed that the N-terminal region encompassing amino acids 1 to 125, which is shared in all UL112-113 proteins and highly conserved among betaherpesviruses, is required for both self-interaction and nuclear localization as foci. Further localization studies revealed that, unlike the 43-, 50-, and 84-kDa proteins, which were distributed as nuclear punctate forms, the 34-kDa form was located predominantly in the cytoplasm. However, when all four proteins were coexpressed simultaneously, all of the UL112-113 proteins were efficiently localized to the promyelocytic leukemia oncogenic domains. We also found that the ability of the UL112-113 proteins to relocate UL44 (the viral polymerase processivity factor) to prereplication foci relied on self-interaction and reached maximal levels when the four proteins were coexpressed. Therefore, our data suggest that interactions occurring among UL112-113 proteins via their shared N-terminal regions are important to both their intranuclear targeting and the recruitment of UL44 to subnuclear sites for viral replication.

Composition of Pseudorabies Virus Particles Lacking Tegument Protein US3, UL47, or UL49 or Envelope Glycoprotein E

Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.

Herpes Simplex Virus Type 1 Infection Induces Activation and Recruitment of Protein Kinase C to the Nuclear Membrane and Increased Phosphorylation of Lamin B

We report that herpes simplex virus type 1 (HSV-1) infection leads to the recruitment of protein kinase C (PKC) to the nuclear rim. In HEp-2 cells, PKC recruitment to the nuclear rim was initiated between 8 h and 12 h postinfection. PKCdelta, a proapoptotic kinase, was completely recruited to the nuclear rim upon infection with HSV-1. PKCalpha was less dramatically relocalized mostly at the nuclear rim upon infection, although some PKCalpha remained in the cytoplasm. PKCzeta-specific immunofluorescence was not significantly relocated to the nuclear rim. The UL34 and UL31 proteins, as well as their association, were each required for PKC recruitment to the nuclear rim. The HSV-1 US3 protein product, a kinase which regulates the phosphorylation state and localization of UL34, was not required for PKC recruitment to the nuclear rim; however, it was required for proper localization along the nuclear rim, as PKC appeared unevenly distributed along the nuclear rim of cells infected with US3 null and kinase-dead mutants. HSV-1 infection induced the phosphorylation of both lamin B and PKC. Elevated lamin B phosphorylation in HSV-1-infected cells was partially reduced by inhibitors of PKC. The data suggest a model in which kinases that normally disassemble the nuclear lamina during apoptosis are recruited to the nuclear membrane through functions requiring UL31 and UL34. We hypothesize that the recruitment of PKC functions to phosphorylate lamin B to help modify the nuclear lamina and promote budding of nucleocapsids at the inner nuclear membrane.

Identification and Functional Evaluation of Cellular and Viral Factors Involved in the Alteration of Nuclear Architecture during Herpes Simplex Virus 1 Infection

Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.

Identification of Proteins Phosphorylated Directly by the Us3 Protein Kinase Encoded by Herpes Simplex Virus 1

We have developed a system to analyze the specific protein kinase activity of herpes simplex virus 1 Us3 in vitro and shown that Us3 directly phosphorylates viral proteins UL34, ICP22, and Us9 and the cellular protein Bad, previously reported to be putative substrates. Using this system, we determined the phosphorylation sites of UL34 and identified UL31 as a previously unreported, novel substrate of Us3. This system will be useful for further identification of Us3 substrates and their phosphorylation sites, clarification of the role of Us3 in viral replication, and identification of additional Us3 function(s).

Identification of an Essential Domain in the Herpes Simplex Virus 1 U L 34 Protein That Is Necessary and Sufficient To Interact with U L 31 Protein

Previous results have indicated that the herpes simplex virus 1 UL31 and UL34 proteins interact and form a complex at the inner nuclear membranes of infected cells, where both play important roles in the envelopment of nucleocapsids at the inner nuclear membrane. In the work described here, mapping studies using glutathione S-transferase pull-down assays indicated that amino acids 137 to 181 of the UL34 protein are sufficient to mediate an interaction with the UL31 protein. A recombinant virus (v3480) lacking UL34 codons 138 to 181 was constructed. Similar to a UL34 null virus, v3480 failed to replicate on Vero cells and grew to a limited extent on rabbit skin cells. A UL34-expressing cell line restored v3480 growth and plaque formation. Similar to the localization of UL31 protein in cells infected with a UL34 null virus, the UL31 protein was present in the nuclei of Hep2 cells infected with v3480. Hep2 cells infected with v3480 contained the UL34 protein in the cytoplasm, the nucleus, and the nuclear membrane, and this was noted to be similar to the appearance of cells infected with a UL31 null virus. In transient expression assays, the interaction between UL34 amino acids 137 to 181 and the UL31 protein was sufficiently robust to target green fluorescent protein and emerin to intranuclear sites that contained the UL31 protein. These data indicate that amino acids 137 to 181 of the UL34 protein are (i) sufficient to mediate interactions with the UL31 protein in vitro and in vivo, (ii) necessary for the colocalization of UL31 and UL34 in infected cells, and (iii) essential for normal viral replication.

Decrease in Sensitivity of PIG-A-Mutant Cells to Natural Killer Cells Conferred by Missing of Stress-Inducible Membrane Proteins ULBPs.

The mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand to manifest symptoms is still unknown. PNH cells with PIG-A mutations do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of a series of GPI-linked membrane proteins. Blood cells with PNH phenotype (GPI− cells) expand in patients with bone marrow (BM) failure syndromes including PNH, aplastic anemia, and myelodysplastic syndromes. The diseases share an immune-mediated BM injury possibly by cytotoxic lymphocytes such as natural killer (NK) cells and cytotoxic T lymphocytes (CTL). It is suggested that the BM injury allows PNH clones survive selectively. Indeed, we have shown that human leukemic cells (K562) decrease their sensitivity to NK cells in vitro when K562 cells acquire PIG-A mutations (Nagakura et al, Blood 2002). In the present study, we show that the decrease in NK sensitivity of PIG-A mutant cells is ascribable to the deficiency of GPI-linked membrane proteins, ULBPs. ULBPs bind cytomegalovirus protein UL-16, consist of ULBP1-3, and emerge in cell membrane when cells are infected or transformed. They trigger off a NK activation signal, which overrides an inhibitory signal from MHC class I (Cosman et al, Immunity 2001). ULBPs also activate CTL besides NK cells. As target cells, a pair of GPI+ control and GPI− mutant cell lines was prepared from a GPI− K562 cell line bearing a PIG-A mutation by transfection with a PIG-A cDNA and a vector alone, respectively. Flow cytometry detected ULBP1-3 on the surface of GPI+ but none of GPI− K562 cells. As effector cells, we used cultured human NK cells (KHYG-1) deficient in Fc γR type III (CD16). GPI− cells were more resistant than GPI+ cells to the killing by NK cells in the 51Cr-release assay. GPI+ cells decreased their sensitivity to NK cells to the level of GPI− cells in the presence of antibodies to both ULBP1 and ULBP2, while antibodies to ULBP3 exerted no effects. None of the antibodies to ULBPs showed any effects on the killing of GPI− cells, while antibodies to NKG2D, which is a NK receptor for both ULBPs and MICA/B, inhibited the killing of both GPI+ and GPI− cells. Thus, GPI− cells that lack membrane ULBPs show a survival advantage in the setting of immune attack in vitro. Of clinical interests, ULBPs were detected on the cell surface of GPI+ but none of GPI− granulocytes of patients with PNH. GPI+ granulocytes of healthy individuals were negative for ULBPs. There appears pressure to induce membrane ULBPs in patients with PNH, leading to BM injury. These findings suggest that the failure of membrane expression of ULBPs permits selective expansion of PNH clones in patients with PNH and other BM failure syndromes.

Characterization of the human cytomegalovirus UL34 gene.

UL34 encodes the transcriptional repressor of the human cytomegalovirus immune evasion gene, US3, and is essential for viral replication in tissue culture. Two different monocistronic transcripts originate from UL34 at early and late times postinfection and encode two predominant proteins and a third, minor protein. The UL34 proteins are differentially expressed throughout the viral replication cycle, with both proteins localizing to the nucleus and repressing expression of the US3 gene.

Two human ULBP/RAET1 molecules with transmembrane regions are ligands for NKG2D.

We characterized two novel members of the RAET1/ULBP gene cluster, RAET1E and RAET1G. The encoded proteins were similar to the ULBP in their class I-like alpha1 and alpha2 domains, but differed in that, instead of being GPI-anchored, their sequences were type 1 membrane-spanning molecules. Both proteins were capable of being expressed at the cell surface. Both proteins bound the activating receptor NKG2D, and RAET1G bound the human CMV protein UL16. The expression of diverse NKG2D-binding molecules in different tissues and with different properties is consistent with multiple modes of infection- or stress-induced activation.

Conformational changes in the nuclear lamina induced by herpes simplex virus type 1 require genes U(L)31 and U(L)34.

The herpes simplex virus type 1 (HSV-1) U(L)31 and U(L)34 proteins are dependent on each other for proper targeting to the nuclear membrane and are required for efficient envelopment of nucleocapsids at the inner nuclear membrane. In this work, we show that whereas the solubility of lamins A and C (lamin A/C) was not markedly increased, HSV induced conformational changes in the nuclear lamina of infected cells, as viewed after staining with three different lamin A/C-specific antibodies. In one case, reactivity with a monoclonal antibody that recognizes an epitope in the lamin tail domain was greatly reduced in HSV-infected cells. This apparent HSV-induced epitope masking required both U(L)31 and U(L)34, but these proteins were not sufficient to mask the epitope in uninfected cells, indicating that other HSV proteins are also required. In the second case, staining with a rabbit polyclonal antibody that primarily recognizes epitopes in the lamin A/C rod domain revealed that U(L)34 is required for HSV-induced decreased availability of epitopes for reaction with the antibody, whereas U(L)31 protein was dispensable for this effect. Still another polyclonal antibody indicated virtually no difference in lamin A/C staining in infected versus uninfected cells, indicating that the HSV-induced changes are more conformational than the result of lamin depletion at the nuclear rim. Further evidence supporting an interaction between the nuclear lamina and the U(L)31/U(L)34 protein complex includes the observations that (i) overexpression of the U(L)31 protein in uninfected cells was sufficient to relocalize lamin A/C from the nuclear rim into nucleoplasmic aggregates, (ii) overexpression of U(L)34 was sufficient to relocalize some lamin A/C into the cytoplasm, and (iii) both U(L)31 and U(L)34 could directly bind lamin A/C in vitro. These studies suggest that the U(L)31 and U(L)34 proteins modify the conformation of the nuclear lamina in infected cells, possibly by direct interaction with lamin A/C, and that other proteins are also likely involved. Given that the nuclear lamina potentially excludes nucleocapsids from envelopment sites at the inner nuclear membrane, the lamina alteration may reflect a role of the U(L)31/U(L)34 protein complex in perturbing the lamina to promote nucleocapsid egress from the nucleus. Alternatively, the data are compatible with a role of the lamina in targeting the U(L)31/U(L)34 protein complex to the nuclear membrane.

Herpes simplex virus type 1 primary envelopment: UL34 protein modification and the US3-UL34 catalytic relationship.

The herpes simplex virus type 1 (HSV-1) US3 kinase is likely important for primary envelopment of progeny nucleocapsids since it localizes to the nuclear envelope of infected cells and largely determines the phosphorylation state and localization of the necessary primary envelopment factor, the UL34 protein. In HEp-2 cells, the production of infectious US3 null progeny is delayed and decreased relative to that of the parental strain, HSV-1(F). Furthermore, the US3 kinase affects the morphology of primary envelopment such that in its absence, UL34 protein-containing enveloped virions accumulate within membrane-bound vesicles. These vesicles are most often found along the interior periphery of the nucleus and may be derived from the inner nuclear membrane. Since the US3 and UL34 proteins comprise a kinase-substrate pair, a reasonable hypothesis is that the US3 kinase influences these replication parameters by direct phosphorylation of the UL34 protein. For this report, recombinant viruses were constructed to determine the significance of UL34 protein phosphorylation and US3 catalytic activity on UL34 protein localization, single-step growth, and envelopment morphology in both HEp-2 and Vero cells. The data presented suggest that the significance of UL34 phosphorylation is cell type dependent and that efficient viral morphogenesis requires US3-mediated phosphorylation of an infected cell protein other than UL34.

Herpes simplex virus type 1 portal protein UL6 interacts with the putative terminase subunits UL15 and UL28.

The herpes simplex virus type 1 (HSV-1) UL6, UL15, and UL28 proteins are essential for cleavage of replicated concatemeric viral DNA into unit length genomes and their packaging into a preformed icosahedral capsid known as the procapsid. The capsid-associated UL6 DNA-packaging protein is located at a single vertex and is thought to form the portal through which the genome enters the procapsid. The UL15 protein interacts with the UL28 protein, and both are strong candidates for subunits of the viral terminase, a key component of the molecular motor that drives the DNA into the capsid. To investigate the association of the UL6 protein with the UL15 and UL28 proteins, the three proteins were produced in large amounts in insect cells with the baculovirus expression system. Interactions between UL6 and UL28 and between UL6 and UL15 were identified by an immunoprecipitation assay. These results were confirmed by transiently expressing wild-type and mutant proteins in mammalian cells and monitoring their distribution by immunofluorescence. In cells expressing the single proteins, UL6 and UL15 were concentrated in the nuclei whereas UL28 was found in the cytoplasm. When the UL6 and UL28 proteins were coexpressed, UL28 was redistributed to the nuclei, where it colocalized with UL6. In cells producing either of two cytoplasmic UL6 mutant proteins and a functional epitope-tagged form of UL15, the UL15 protein was concentrated with the mutant UL6 protein in the cytoplasm. These observed interactions of UL6 with UL15 and UL28 are likely to be of major importance in establishing a functional DNA-packaging complex at the portal vertex of the HSV-1 capsid.

The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins.

Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.