Differing Roles of Inner Tegument Proteins pUL36 and pUL37 during Entry of Herpes Simplex Virus Type 1

ABSTRACT Studies with herpes simplex virus type 1 (HSV-1) have shown that secondary envelopment and virus release are blocked in mutants deleted for the tegument protein gene UL36 or UL37, leading to the accumulation of DNA-containing capsids in the cytoplasm of infected cells. The failure to assemble infectious virions has meant that the roles of these genes in the initial stages of infection could not be investigated. To circumvent this, cells infected at a low multiplicity were fused to form syncytia, thereby allowing capsids released from infected nuclei access to uninfected nuclei without having to cross a plasma membrane. Visualization of virus DNA replication showed that a UL37-minus mutant was capable of transmitting infection to all the nuclei within a syncytium as efficiently as the wild-type HSV-1 strain 17+ did, whereas infection by UL36-minus mutants failed to spread. Thus, these inner tegument proteins have differing functions, with pUL36 being essential during both the assembly and uptake stages of infection, while pUL37 is needed for the formation of virions but is not required during the initial stages of infection. Analysis of noninfectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.

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UL36p Is Required for Efficient Transport of Membrane-Associated Herpes Simplex Virus Type 1 along Microtubules

Journal of Virology ◽

10.1128/jvi.00225-08 ◽

2008 ◽

Vol 82 (15) ◽

pp. 7388-7394 ◽

Cited By ~ 52

Author(s):

Sara K. Shanda ◽

Duncan W. Wilson

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Anterograde Transport ◽

Tegument Proteins ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Microtubule-mediated anterograde transport is essential for the transport of herpes simplex virus type 1 (HSV-1) along axons, yet little is known regarding the mechanism and the machinery required for this process. Previously, we were able to reconstitute anterograde transport of HSV-1 on microtubules in an in vitro microchamber assay. Here we report that the large tegument protein UL36p is essential for this trafficking. Using a fluorescently labeled UL36 null HSV-1 strain, KΔUL36GFP, we found that it is possible to isolate a membrane-associated population of this virus. Although these viral particles contained normal amounts of tegument proteins VP16, vhs, and VP22, they displayed a 3-log decrease in infectivity and showed a different morphology compared to UL36p-containing virions. Membrane-associated KΔUL36GFP also displayed a slightly decreased binding to microtubules in our microchamber assay and a two-thirds decrease in the frequency of motility. This decrease in binding and motility was restored when UL36p was supplied in trans by a complementing cell line. These findings suggest that UL36p is necessary for HSV-1 anterograde transport.

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Suppression of Proinflammatory Cytokine Expression by Herpes Simplex Virus Type 1

Journal of Virology ◽

10.1128/jvi.78.11.5883-5890.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5883-5890 ◽

Cited By ~ 51

Author(s):

Trine H. Mogensen ◽

Jesper Melchjorsen ◽

Lene Malmgaard ◽

Antonella Casola ◽

Søren R. Paludan

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Proinflammatory Cytokines ◽

Herpes Simplex Virus Type ◽

Cytokine Expression ◽

Wild Type ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Viral immune evasion strategies are important for establishment and maintenance of infections. Many viruses are in possession of mechanisms to counteract the antiviral response raised by the infected host. Here we show that a herpes simplex virus type 1 (HSV-1) mutant lacking functional viral protein 16 (VP16)—a tegument protein promoting viral gene expression—induced significantly higher levels of proinflammatory cytokines than wild-type HSV-1. This was observed in several cell lines and primary murine macrophages, as well as in peritoneal cells harvested from mice infected in vivo. The enhanced ability to stimulate cytokine expression in the absence of VP16 was not mediated directly by VP16 but was dependent on the viral immediate-early genes for infected cell protein 4 (ICP4) and ICP27, which are expressed in a VP16-dependent manner during primary HSV infection. The virus appeared to target cellular factors other than interferon-induced double-stranded RNA-activated protein kinase R (PKR), since the virus mutants remained stronger inducers of cytokines in cells stably expressing a dominant-negative mutant form of PKR. Finally, mRNA stability assay revealed a significantly longer half-life for interleukin-6 mRNA after infection with the VP16 mutant than after infection with the wild-type virus. Thus, HSV is able to suppress expression of proinflammatory cytokines by decreasing the stability of mRNAs, thereby potentially impeding the antiviral host response to infection.

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A Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutant with Increased Virulence and Reduced Spontaneous Reactivation

Journal of Virology ◽

10.1128/jvi.73.2.920-929.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 920-929 ◽

Cited By ~ 40

Author(s):

Guey-Chuen Perng ◽

Susan M. Slanina ◽

Ada Yukht ◽

Barbara S. Drolet ◽

William Keleher ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Ocular Infection ◽

Wild Type ◽

Simplex Virus ◽

Spontaneous Reactivation ◽

Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for efficient spontaneous reactivation of HSV-1 from latency. We previously reported that insertion of the LAT promoter and just the first 1.5 kb of the 8.3-kb LAT gene into an ectopic location in the virus restored wild-type spontaneous reactivation to a LAT null mutant. This mutant, LAT3.3A (previously designated LAT1.5a), thus showed that the expression of just the first 1.5 kb of LAT is sufficient for wild-type spontaneous reactivation. We also showed that in the context of the entire LAT gene, deletion of LAT nucleotides 76 to 447 (LAT mutantdLAT371) had no effect on spontaneous reactivation or virulence. We report here on a LAT mutant designated LAT2.9A. This mutant is similar to LAT3.3A, except that the ectopic LAT insert contains the same 371-nucleotide deletion found in dLAT371. We found that LAT2.9A had a significantly reduced rate of spontaneous reactivation compared to marker-rescued and wild-type viruses. This was unexpected, since the combined results of dLAT371 and LAT3.3A predicted that spontaneous reactivation of LAT2.9A would be wild type. We also found that LAT2.9A was more virulent than wild-type or marker-rescued viruses after ocular infection of rabbits. This was unexpected, since LAT null mutants and LAT3.3A have wild-type virulence. These results suggest for the first time (i) that regions past the first 1.5 kb of LAT can compensate for deletions in the first 1.5kb of LAT and may therefore play a role in LAT dependent spontaneous reactivation and (ii) that regions of LAT affect viral virulence.

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The Herpes Simplex Virus Type 1 Cleavage/Packaging Protein, UL32, Is Involved in Efficient Localization of Capsids to Replication Compartments

Journal of Virology ◽

10.1128/jvi.72.3.2463-2473.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 2463-2473 ◽

Cited By ~ 92

Author(s):

Carmela Lamberti ◽

Sandra K. Weller

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Nuclear Staining ◽

Wild Type ◽

Viral Dna ◽

Infected Cells ◽

Simplex Virus

ABSTRACT Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant, tsN20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57–71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938–3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in “bringing” preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.

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Scopadulciol is an Inhibitor of Herpes Simplex Virus Type 1 and a Potentiator of Aciclovir

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029600700204 ◽

1996 ◽

Vol 7 (2) ◽

pp. 79-85 ◽

Cited By ~ 11

Author(s):

Kyoko Hayashi ◽

Toshimitsu Hayashi

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Antiviral Activity ◽

Virus Type ◽

Hela Cells ◽

Herpes Simplex Virus Type ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

The antiviral activity of scopadulciol (SDC), a tetracyclic diterpenoid with a chemical structure related to that of aphidicolin, isolated from Scoparia dulcis, was studied in vitro against herpes simplex virus type 1 (HSV-1). SDC was found to inhibit the virus replication as shown by reduction of virus production. The action was not due to the inhibition of viral DNA polymerase activity and virus penetration, but might involve, at least in part, a virucidal effect. SDC did not suppress the viral protein synthesis of infected cells when added at an early stage of HSV-1 replication, but did when added later. When aciclovir (ACV) and SDC were evaluated in combination for antiviral activity against HSV-1 replication and cytotoxicity, these drugs inhibited viral replication in HeLa cells synergistically, but the same combination did not produce synergistic cytotoxicity in HeLa cells. Studies of the deoxynucleotide pool sizes revealed that SDC increased the intracellular dNTP pools and ACV triphosphate level significantly in infected cells when the cells were treated with the combination. These results could account for the synergistic action between SDC and ACV.

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Membrane Association of VP22, a Herpes Simplex Virus Type 1 Tegument Protein

Journal of Virology ◽

10.1128/jvi.77.8.4888-4898.2003 ◽

2003 ◽

Vol 77 (8) ◽

pp. 4888-4898 ◽

Cited By ~ 45

Author(s):

Michael J. Brignati ◽

Joshua S. Loomis ◽

John W. Wills ◽

Richard J. Courtney

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Membrane Association ◽

Tegument Proteins ◽

Simplex Virus ◽

Acidic Compartments ◽

Hsv 1

ABSTRACT Tegument proteins of herpes simplex virus type 1 (HSV-1) are hypothesized to contain the functional information required for the budding or envelopment process proposed to occur at cytoplasmic compartments of the host cell. One of the most abundant tegument proteins of HSV-1 is the UL49 gene product, VP22, a 38-kDa protein of unknown function. To study its subcellular localization, a VP22-green fluorescent protein chimera was expressed in transfected human melanoma (A7) cells. In the absence of other HSV-1 proteins, VP22 localizes to acidic compartments of the cell that may include the trans-Golgi network (TGN), suggesting that this protein is membrane associated. Membrane pelleting and membrane flotation assays confirmed that VP22 partitions with the cellular membrane fraction. Through truncation mutagenesis, we determined that the membrane association of VP22 is a property attributed to amino acids 120 to 225 of this 301-amino-acid protein. The above results demonstrate that VP22 contains specific information required for targeting to membranes of acidic compartments of the cell which may be derived from the TGN, suggesting a potential role for VP22 during tegumentation and/or final envelopment.

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Negative regulation of herpes simplex virus type 1 ICP4 promoter by IE180 protein of pseudorabies virus

Journal of General Virology ◽

10.1099/vir.0.80119-0 ◽

2004 ◽

Vol 85 (8) ◽

pp. 2125-2130 ◽

Cited By ~ 12

Author(s):

S. Gómez-Sebastián ◽

E. Tabarés

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Negative Regulation ◽

Wild Type ◽

Recombinant Viruses ◽

Simplex Virus ◽

Hsv 1

Recombinant pseudorabies viruses (PRVs) gIS8 and N1aHTK were constructed by the insertion of a chimeric gene (α4–TK) from herpes simplex virus type 1 (HSV-1) into wild-type PRV. HSV-1 TK expression by these recombinant viruses resulted in enhanced sensitivity to ganciclovir, compared to that of the wild-type PRV, and was similar to the sensitivity shown by HSV-1. Infection with gIS8 or N1aHTK recombinant viruses led to expression of HSV-1 TK mRNA as an immediate–early (IE) gene, observed by downregulation of the HSV-1 α4 promoter. This negative regulation was due to a PRV IE protein, IE180. IE180, however, does not have all the regulatory functions of the infected-cell protein ICP4, as it does not restore the growth of ICP4-deficient HSV-1 mutants.

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Nuclear Localizations of the Herpes Simplex Virus Type 1 Tegument Proteins VP13/14, vhs, and VP16 Precede VP22-Dependent Microtubule Reorganization and VP22 Nuclear Import

Journal of Virology ◽

10.1128/jvi.79.8.4730-4743.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4730-4743 ◽

Cited By ~ 28

Author(s):

Jamie C. Yedowitz ◽

Anna Kotsakis ◽

Elisabeth F. M. Schlegel ◽

John A. Blaho

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Herpes Simplex Virus Type ◽

Full Length ◽

Acetylated Tubulin ◽

Tegument Proteins ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol. 75:8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (UL49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while α-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.

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The Dominant-Negative Herpes Simplex Virus Type 1 (HSV-1) Recombinant CJ83193 Can Serve as an Effective Vaccine against Wild-Type HSV-1 Infection in Mice

Journal of Virology ◽

10.1128/jvi.78.11.5756-5765.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5756-5765 ◽

Cited By ~ 16

Author(s):

Hanka Augustinova ◽

Daniela Hoeller ◽

Feng Yao

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Dominant Negative ◽

Trigeminal Ganglia ◽

Wild Type ◽

Simplex Virus ◽

Hsv 1

ABSTRACT By selectively regulating the expression of the trans-dominant-negative mutant polypeptide UL9-C535C, of herpes simplex virus type 1 (HSV-1) origin binding protein UL9 with the tetracycline repressor (tetR)-mediated gene switch, we recently generated a novel replication-defective and anti-HSV-specific HSV-1 recombinant, CJ83193. The UL9-C535C peptides expressed by CJ83193 can function as a potent intracellular therapy against its own replication, as well as the replication of wild-type HSV-1 and HSV-2 in coinfected cells. In this report, we demonstrate that CJ83193 cannot initiate acute productive infection in corneas of infected mice nor can it reactivate from trigeminal ganglia of mice latently infected by CJ83193 in a mouse ocular model. Given that CJ83193 is capable of expressing the viral α, β, and γ1 genes but little or no γ2 genes, we tested the vaccine potential of CJ83193 against HSV-1 infection in a mouse ocular model. Our studies showed that immunization with CJ83193 significantly reduced the yields of challenge HSV in the eyes and trigeminal ganglia on days 3, 5, and 7 postchallenge. Like in mice immunized with the wild-type HSV-1 strain KOS, immunization of mice with CJ83193 prevents the development of keratitis and encephalitis induced by corneal challenge with wild-type HSV-1 strain mP. Delayed-type hypersensitivity (DTH) assays demonstrate that CJ83193 can elicit durable cell-mediated immunity at the same level as that of wild-type HSV-1 and is more effective than that induced by d27, an HSV-1 ICP27 deletion mutant. Moreover, mice immunized with CJ83193 developed strong, durable HSV-1-neutralizing antibodies at levels at least twofold higher than those induced by d27. The results presented in this report have shed new light on the development of effective HSV viral vaccines that encode a unique safety mechanism capable of inhibiting the mutant's own replication and that of wild-type virus.

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DNA Cleavage and Packaging Proteins Encoded by Genes UL28, UL15, and UL33 of Herpes Simplex Virus Type 1 Form a Complex in Infected Cells

Journal of Virology ◽

10.1128/jvi.76.10.4785-4791.2002 ◽

2002 ◽

Vol 76 (10) ◽

pp. 4785-4791 ◽

Cited By ~ 53

Author(s):

Philippa M. Beard ◽

Naomi S. Taus ◽

Joel D. Baines

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Dna Cleavage ◽

Rabbit Polyclonal Antibody ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Previous studies have indicated that the UL6, UL15, UL17, UL28, UL32, and UL33 genes are required for the cleavage and packaging of herpes simplex viral DNA. To identify proteins that interact with the UL28-encoded DNA binding protein of herpes simplex virus type 1 (HSV-1), a previously undescribed rabbit polyclonal antibody directed against the UL28 protein fused to glutathione S-transferase was used to immunopurify UL28 and the proteins with which it associated. It was found that the antibody specifically coimmunoprecipitated proteins encoded by the genes UL28, UL15, and UL33 from lysates of both HEp-2 cells infected with HSV-1(F) and insect cells infected with recombinant baculoviruses expressing these three proteins. In reciprocal reactions, antibodies directed against the UL15- or UL33-encoded proteins also coimmunoprecipitated the UL28 protein. The coimmunoprecipitation of the three proteins from HSV-infected cells confirms earlier reports of an association between the UL28 and UL15 proteins and represents the first evidence of the involvement of the UL33 protein in this complex.

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