The pseudorabies virus VP1/2 tegument protein is required for intracellular capsid transport

Transport of capsids in cells is critical to alphaherpesvirus infection and pathogenesis; however, viral factors required for transport have yet to be identified. Here we provide a detailed examination of capsid dynamics during the egress phase of infection in Vero cells infected with pseudorabies virus. We demonstrate that the VP1/2 tegument protein is required for processive microtubule-based transport of capsids in the cytoplasm. A second tegument protein that binds to VP1/2, UL37, was necessary for wild-type transport but was not essential for this process. Both proteins were also required for efficient nuclear egress of capsids to the cytoplasm.     

Improper Tagging of the Non-Essential Small Capsid Protein VP26 Impairs Nuclear Capsid Egress of Herpes Simplex Virus

To analyze the subcellular trafficking of herpesvirus capsids, the small capsid protein has been labeled with different fluorescent proteins. Here, we analyzed the infectivity of several HSV1(17(+)) strains in which the N-terminal region of the non-essential small capsid protein VP26 had been tagged at different positions. While some variants replicated with similar kinetics as their parental wild type strain, others were not infectious at all. Improper tagging resulted in the aggregation of VP26 in the nucleus, prevented efficient nuclear egress of viral capsids, and thus virion formation. Correlative fluorescence and electron microscopy showed that these aggregates had sequestered several other viral proteins, but often did not contain viral capsids. The propensity for aggregate formation was influenced by the type of the fluorescent protein domain, the position of the inserted tag, the cell type, and the progression of infection. Among the tags that we have tested, mRFPVP26 had the lowest tendency to induce nuclear aggregates, and showed the least reduction in replication when compared to wild type. Our data suggest that bona fide monomeric fluorescent protein tags have less impact on proper assembly of HSV1 capsids and nuclear capsid egress than tags that tend to dimerize. Small chemical compounds capable of inducing aggregate formation of VP26 may lead to new antiviral drugs against HSV infections.     

BFRF1 protein is involved in EBV-mediated autophagy manipulation

Viral egress and autophagy are two mechanisms that seem to be strictly connected in Herpesviruses’s biology. Several data suggest that the autophagic machinery facilitates the egress of viral capsids and thus the production of new infectious particles. In the Herpesvirus family, viral nuclear egress is controlled and organized by a well conserved group of proteins named Nuclear Egress Complex (NEC). In the case of EBV, NEC is composed by BFRF1 and BFLF2 proteins, although the alterations of the nuclear host cell architecture are mainly driven by BFRF1, a multifunctional viral protein anchored to the inner nuclear membrane of the host cell. BFRF1 shares a peculiar distribution with several nuclear components and with them it strictly interacts. In this study, we investigated the possible role of BFRF1 in manipulating autophagy, pathway that possibly originates from nucleus, regulating the interplay between autophagy and viral egress.     

A physical link between the pseudorabies virus capsid and the nuclear egress complex

Following their assembly, herpesvirus capsids exit the nucleus by budding at the inner nuclear membrane. Two highly conserved viral proteins are required for this process, pUL31 and pUL34. In this report, we demonstrate that the pUL31 component of the pseudorabies virus nuclear egress complex is a conditional capsid-binding protein that is unmasked in the absence of pUL34. The interaction between pUL31 and capsids was confirmed through fluorescence microscopy and Western blot analysis of purified intranuclear capsids. Three viral proteins were tested for their abilities to mediate the pUL31-capsid interaction: the minor capsid protein pUL25, the portal protein pUL6, and the terminase subunit pUL33. Despite the requirement for each protein in nuclear egress, none of these viral proteins were required for the pUL31-capsid interaction. These findings provide the first formal evidence that a herpesvirus nuclear egress complex interacts with capsids and have implications for how DNA-containing capsids are selectively targeted for nuclear egress.     

Impairment of nuclear pores in bovine herpesvirus 1-infected MDBK cells

Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.     

Nuclear egress and envelopment of herpes simplex virus capsids analyzed with dual-color fluorescence HSV1(17+)

To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as β-galactosidase expression cassettes. When the BAC pHSV1(17⁺)blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17⁺)blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17⁺)blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.     

Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus

Common features of parvovirus capsids are open pores at the fivefold symmetry axes that traverse the virion shell. Upon limited heat treatment in vitro, the pores can function as portals to externalize VP1/VP2 protein N-terminal sequences which harbor infection-relevant functional domains, such as a phospholipase A(2) catalytic domain. Here we show that adeno-associated virus type 2 (AAV2) also exposes its VP1/VP2 N termini in vivo during infection, presumably in the endosomal compartment. This conformational change is influenced by treatment with lysosomotropic reagents. While incubation of cells with bafilomycin A1 reduced exposure of VP1/VP2 N termini, incubation with chloroquine stimulated externalization transiently. N-terminally located basic amino acid clusters with nuclear localization activity also become exposed in this process and are accessible on the virus capsid when it enters the cytoplasm. This is an obligatory step in AAV2 infection. However, a direct role of these sequences in nuclear translocation of viral capsids could not be determined by microinjection of wild-type or mutant viruses. This suggests that further modifications of the capsid have to take place in a precytoplasmic entry step that prepares the virus for nuclear entry. Microinjection of several capsid-specific antibodies into the cell nucleus blocked AAV2 infection completely, supporting the conclusion that AAV2 capsids bring the infectious genome into the nucleus.     

Eclipse phase of herpes simplex virus type 1 infection: Efficient dynein-mediated capsid transport without the small capsid protein VP26

Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-DeltaVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-DeltaVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-DeltaVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin.     

In rat dorsal root ganglion neurons,herpes simplex virus type 1 tegument forms in the cytoplasm of the cell body

The herpes simplex virus type 1 (HSV-1) tegument is the least understood component of the virion, and the mechanism of tegument assembly and incorporation into virions during viral egress has not yet been elucidated. In the present study, the addition of tegument proteins (VP13/14, VP16, VP22, and US9) and envelope glycoproteins (gD and gH) to herpes simplex virions in the cell body of rat dorsal root ganglion neurons was examined by immunoelectron microscopy. All tegument proteins were detected diffusely spread in the nucleus within 10 to 12 h and, at these times, nucleocapsids were observed budding from the nucleus. The majority (96%) of these nucleocapsids had no detectable label for tegument and glycoproteins despite the presence of tegument proteins in the nucleus and glycoproteins adjacent to the nuclear membrane. Immunolabeling for tegument proteins and glycoproteins was found abundantly in the cytoplasm of the cell body in multiple discrete vesicular areas: on unenveloped, enveloped, or partially enveloped capsids adjacent to these vesicles and in extracellular virions. These vesicles and intracytoplasmic and extracellular virions also labeled with Golgi markers, giantin, mannosidase II, and TGN38. Treatment with brefeldin A from 2 to 24 h postinfection markedly inhibited incorporation into virions of VP22 and US9 but to a lesser degree with VP16 and VP13/14. These results suggest that, in the cell body of neurons, most tegument proteins are incorporated into unenveloped nucleocapsids prior to envelopment in the Golgi and the trans-Golgi network. These findings give further support to the deenvelopment-reenvelopment hypothesis for viral egress. Finally, the addition of tegument proteins to unenveloped nucleocapsids in the cell body allows access to these unenveloped nucleocapsids to one of two pathways: egress through the cell body or transport into the axon.     

Cytomegalovirus pUL96 is critical for the stability of pp150-associated nucleocapsids

Maturation of human cytomegalovirus (HCMV) initiates with nucleocapsids that egress from the nucleus and associate with a juxtanuclear cytoplasmic assembly compartment, where virion envelopment and release are orchestrated. Betaherpesvirus conserved proteins pp150 (encoded by UL32) and pUL96 are critical for HCMV growth in cell culture. pp150 is a capsid-proximal tegument protein that preserves the integrity of nucleocapsids during maturation. pUL96, although expressed as an early protein, acts late during virus maturation, similar to pp150, based on the comparable antigen distribution in UL96, UL32, or UL96/UL32 dual mutant virus-infected cells. pp150 associates with nuclear capsids prior to DNA encapsidation, whereas both pp150 and pUL96 associate with extracellular virus, suggesting that pUL96 is added after pp150. In the absence of pUL96, capsid egress from the nucleus continues; however, unlike wild-type virus infection, pp150 accumulates in the nuclear, as well as in the cytoplasmic, compartment. Ultrastructural evaluation of a UL96 conditional mutant revealed intact nuclear stages but aberrant nucleocapsids accumulating in the cytoplasm comparable to the known phenotype of UL32 mutant virus. In summary, pUL96 preserves the integrity of pp150-associated nucleocapsids during translocation from the nucleus to the cytoplasm.     

Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport

Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model. A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system. A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes. Recombinant herpes simplex virus capsids were constructed either with or without VP26. In pull-down assays VP26+ capsids bound to RP3; VP26-capsids did not. To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37 degrees C. After 1 h VP26+ capsids were observed to co-localize with RP3, Tctex1, and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, whereas VP26-capsids remained in a random distribution. We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains.     

The capsid and tegument of the alphaherpesviruses are linked by an interaction between the UL25 and VP1/2 proteins

How alphaherpesvirus capsids acquire tegument proteins remains a key question in viral assembly. Using pseudorabies virus (PRV), we have previously shown that the 62 carboxy-terminal amino acids of the VP1/2 large tegument protein are essential for viral propagation and when transiently expressed as a fusion to green fluorescent protein relocalize to nuclear capsid assemblons following viral infection. Here, we show that localization of the VP1/2 capsid-binding domain (VP1/2cbd) into assemblons is conserved in herpes simplex virus type 1 (HSV-1) and that this recruitment is specifically on capsids. Using a mutant virus screen, we find that the protein product of the UL25 gene is essential for VP1/2cbd association with capsids. An interaction between UL25 and VP1/2 was corroborated by coimmunoprecipitation from cells transiently expressing either HSV-1 or PRV proteins. Taken together, these findings suggest that the essential function of the VP1/2 carboxy terminus is to anchor the VP1/2 tegument protein to capsids. Furthermore, UL25 encodes a multifunctional capsid protein involved in not only encapsidation, as previously described, but also tegumentation.     

Proteolytic cleavage of VP1-2 is required for release of herpes simplex virus 1 DNA into the nucleus

In this report we propose a model in which after the herpes simplex virus (HSV) capsid docks at the nuclear pore, the tegument protein attached to the capsid must be cleaved by a serine or a cysteine protease in order for the DNA to be released into the nucleus. In support of the model are the following results. (i) Exposure of cells at the time of or before infection to l-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK), a serine-cysteine protease inhibitor, prevents the release of viral DNA or expression of viral genes. TPCK does not block viral gene expression after entry of viral DNA into the nucleus. (ii) The tegument protein VP1-2, the product of the U(L)36 gene, is cleaved shortly after the entry of the HSV 1 (HSV-1) virion into the cell. (iii) The proteolytic cleavage of VP1-2 does not occur in cells that are infected with HSV-1 under conditions that prevent the release of the viral DNA into the nucleus. (iv) The proteolytic cleavage of VP1-2 occurs only after the capsid is attached to the nuclear pore. Thus, TPCK prevented the release of HSV-1 DNA into the nucleus when added to medium 1 hour after infection with tsB7 at 39.5 degrees C followed by a shift down to the permissive temperature. The ts lesion maps in the U(L)36 gene. At the nonpermissive temperature, the capsids accumulate at the nuclear pore but the DNA is not released into the nucleus.     

Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

Replication of herpes simplex virus type 1 (HSV-1) involves a step in which a parental capsid docks onto a host nuclear pore complex (NPC). The viral genome then translocates through the nuclear pore into the nucleoplasm, where it is transcribed and replicated to propagate infection. We investigated the roles of viral and cellular proteins in the process of capsid-nucleus attachment. Vero cells were preloaded with antibodies specific for proteins of interest and infected with HSV-1 containing a green fluorescent protein-labeled capsid, and capsids bound to the nuclear surface were quantified by fluorescence microscopy. Results showed that nuclear capsid attachment was attenuated by antibodies specific for the viral tegument protein VP1/2 (UL36 gene) but not by similar antibodies specific for UL37 (a tegument protein), the major capsid protein (VP5), or VP23 (a minor capsid protein). Similar studies with antibodies specific for nucleoporins demonstrated attenuation by antibodies specific for Nup358 but not Nup214. The role of nucleoporins was further investigated with the use of small interfering RNA (siRNA). Capsid attachment to the nucleus was attenuated in cells treated with siRNA specific for either Nup214 or Nup358 but not TPR. The results are interpreted to suggest that VP1/2 is involved in specific attachment to the NPC and/or in migration of capsids to the nuclear surface. Capsids are suggested to attach to the NPC by way of the complex of Nup358 and Nup214, with high-resolution immunofluorescence studies favoring binding to Nup358.     

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.     

A null mutation in the gene encoding the herpes simplex virus type 1 UL37 polypeptide abrogates virus maturation

The tegument is an integral and essential structural component of the herpes simplex virus type 1 (HSV-1) virion. The UL37 open reading frame of HSV-1 encodes a 120-kDa virion polypeptide which is a resident of the tegument. To analyze the function of the UL37-encoded polypeptide a null mutation was generated in the gene encoding this protein. In order to propagate this mutant virus, transformed cell lines that express the UL37 gene product in trans were produced. The null mutation was transferred into the virus genome using these complementing cell lines. A mutant virus designated KDeltaUL37 was isolated based on its ability to form plaques on the complementing cell line but not on nonpermissive (noncomplementing) Vero cells. This virus was unable to grow in Vero cells; therefore, UL37 encodes an essential function of the virus. The mutant virus KDeltaUL37 produced capsids containing DNA as judged by sedimentation analysis of extracts derived from infected Vero cells. Therefore, the UL37 gene product is not required for DNA cleavage or packaging. The UL37 mutant capsids were tagged with the smallest capsid protein, VP26, fused to green fluorescent protein. This fusion protein decorates the capsid shell and consequently the location of the capsid and the virus particle can be visualized in living cells. Late in infection, KDeltaUL37 capsids were observed to accumulate at the periphery of the nucleus as judged by the concentration of fluorescence around this organelle. Fluorescence was also observed in the cytoplasm in large puncta. Fluorescence at the plasma membrane, which indicated maturation and egress of virions, was observed in wild-type-infected cells but was absent in KDeltaUL37-infected cells. Ultrastructural analysis of thin sections of infected cells revealed clusters of DNA-containing capsids in the proximity of the inner nuclear membrane. Occasionally enveloped capsids were observed between the inner and outer nuclear membranes. Clusters of unenveloped capsids were also observed in the cytoplasm of KDeltaUL37-infected cells. Enveloped virions, which were observed in the cytoplasm of wild-type-infected cells, were never detected in the cytoplasm of KDeltaUL37-infected cells. Crude cell fractionation of infected cells using detergent lysis demonstrated that two-thirds of the UL37 mutant particles were associated with the nuclear fraction, unlike wild-type particles, which were predominantly in the cytoplasmic fraction. These data suggest that in the absence of UL37, the exit of capsids from the nucleus is slowed. UL37 mutant particles can participate in the initial envelopment at the nuclear membrane, although this process may be impaired in the absence of UL37. Furthermore, the naked capsids deposited in the cytoplasm are unable to progress further in the morphogenesis pathway, which suggests that UL37 is also required for egress and reenvelopment. Therefore, the UL37 gene product plays a key role in the early stages of the maturation pathway that give rise to an infectious virion.     

Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking

To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry.     

The human cytomegalovirus UL97 protein kinase,an antiviral drug target,is required at the stage of nuclear egress

Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, maribavir. We used maribavir and a UL97 null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNA-containing capsids, in the nuclei of wild-type- and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.     

Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein

VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.     

The UL14 tegument protein of herpes simplex virus type 1 is required for efficient nuclear transport of the alpha transinducing factor VP16 and viral capsids

The protein encoded by the UL14 gene of herpes simplex virus type 1 (HSV-1) and HSV-2 is expressed late in infection and is a minor component of the virion tegument. An UL14-deficient HSV-1 mutant (UL14D) forms small plaques and exhibits an extended growth cycle at low multiplicities of infection (MOI) compared to wild-type virus. Although UL14 is likely to be involved in the process of viral maturation and egress, its precise role in viral replication is still enigmatic. In this study, we found that immediate-early viral mRNA expression was decreased in UL14D-infected cells. Transient coexpression of UL14 and VP16 in the absence of infection stimulated the nuclear accumulation of both proteins. We intended to visualize the fate of VP16 released from the infected virion and constructed UL14-null (14D-VP16G) and rescued (14R-VP16G) viruses that expressed a VP16-green fluorescent protein (GFP) fusion protein. Synchronous high-multiplicity infection of the viruses was performed at 4°C in the absence of de novo protein synthesis. We found that the presence of UL14 in the virion had an enhancing effect on the nuclear accumulation of VP16-GFP. The lack of UL14 did not significantly alter virus internalization but affected incoming capsid transport to the nuclear pore. These observations suggested that UL14 (i) enhanced VP16 nuclear localization at the immediately early phase, thus indirectly regulating the expression of immediate-early genes, and (ii) was associated with efficient nuclear targeting of capsids. The tegument protein UL14 could be part of the machinery that regulates HSV-1 replication.