scholarly journals Nucleolin Is Required for Efficient Nuclear Egress of Herpes Simplex Virus Type 1 Nucleocapsids

2009 ◽  
Vol 84 (4) ◽  
pp. 2110-2121 ◽  
Author(s):  
Ken Sagou ◽  
Masashi Uema ◽  
Yasushi Kawaguchi
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Nuclear Egress ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpesvirus nucleocapsids assemble in the nucleus and must cross the nuclear membrane for final assembly and maturation to form infectious progeny virions in the cytoplasm. It has been proposed that nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane, and these enveloped nucleocapsids then fuse with the outer nuclear membrane to enter the cytoplasm. Little is known about the mechanism(s) for nuclear egress of herpesvirus nucleocapsids and, in particular, which, if any, cellular proteins are involved in the nuclear egress pathway. UL12 is an alkaline nuclease encoded by herpes simplex virus type 1 (HSV-1) and has been suggested to be involved in viral DNA maturation and nuclear egress of nucleocapsids. Using a live-cell imaging system to study cells infected by a recombinant HSV-1 expressing UL12 fused to a fluorescent protein, we observed the previously unreported nucleolar localization of UL12 in live infected cells and, using coimmunoprecipitation analyses, showed that UL12 formed a complex with nucleolin, a nucleolus marker, in infected cells. Knockdown of nucleolin in HSV-1-infected cells reduced capsid accumulation, as well as the amount of viral DNA resistant to staphylococcal nuclease in the cytoplasm, which represented encapsidated viral DNA, but had little effect on these viral components in the nucleus. These results indicated that nucleolin is a cellular factor required for efficient nuclear egress of HSV-1 nucleocapsids in infected cells.

scholarly journals The Conserved Carboxyl-Terminal Half of Herpes Simplex Virus Type 1 Regulatory Protein ICP27 Is Dispensable for Viral Growth in the Presence of Compensatory Mutations

2000 ◽  
Vol 74 (16) ◽  
pp. 7362-7374 ◽  
Author(s):  
Scott M. Bunnell ◽  
Stephen A. Rice
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Vero Cells ◽  
Terminal Portion ◽  
Viral Dna ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that regulates viral gene expression by poorly characterized mechanisms. Previous data suggest that its carboxyl (C)-terminal portion is absolutely required for productive viral infection. In this study, we isolated M16R, a second-site revertant of a viral ICP27 C-terminal mutant. M16R harbors an intragenic reversion, as demonstrated by the fact that its cloned ICP27 allele can complement the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing demonstrated that the intragenic reversion is a frameshift alteration in a homopolymeric run of C residues at codons 215 to 217. This results in the predicted expression of a truncated, 289-residue molecule bearing 72 novel C-terminal residues derived from the +1 reading frame. Consistent with this, M16R expresses an ICP27-related molecule of the predicted size in the nuclei of infected cells. Transfection-based viral complementation assays confirmed that the truncated, frameshifted protein can partially substitute for ICP27 in the context of viral infection. Surprisingly, its novel C-terminal residues are required for this activity. To see if the frameshift mutation is all that is required for M16R's viability, we re-engineered the M16R ICP27 allele and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant, exCd305, was constructed which possesses the frameshift in the context of an ICP27 gene with the C terminus deleted. We found that both M16exC and exCd305 are nonviable in Vero cells, suggesting that one or more extragenic mutations are also required for the viability of M16R. Consistent with this interpretation, we isolated two viable derivatives ofexCd305 which grow productively in Vero cells despite being incapable of encoding the C-terminal portion of ICP27. Studies of viral DNA synthesis in mutant-infected cells indicated that the truncated, frameshifted ICP27 protein can enhance viral DNA replication. In summary, our results demonstrate that the C-terminal portion of ICP27, conserved widely in herpesviruses and previously believed to be absolutely essential, is dispensable for HSV-1 lytic replication in the presence of compensatory genomic mutations.

scholarly journals Host Vesicle Fusion Protein VAPB Contributes to the Nuclear Egress Stage of Herpes Simplex Virus Type-1 (HSV-1) Replication

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 120 ◽  
Author(s):  
Natalia Saiz-Ros ◽  
Rafal Czapiewski ◽  
Ilaria Epifano ◽  
Andrew Stevenson ◽  
Selene Swanson ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Nuclear Membrane ◽  
Herpes Simplex Virus Type ◽  
Vesicle Fusion ◽  
Nuclear Egress ◽  
Simplex Virus ◽  
Hsv 1

The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.

scholarly journals Accumulation of Herpes Simplex Virus Type 1 Early and Leaky-Late Proteins Correlates with Apoptosis Prevention in Infected Human HEp-2 Cells

2001 ◽  
Vol 75 (2) ◽  
pp. 1013-1030 ◽  
Author(s):  
Martine Aubert ◽  
Stephen A. Rice ◽  
John A. Blaho
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Amino Terminal ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803–2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5,d1-2, M11, M15, M16, n504R,n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3,d3-4, d4-5, d5-6, andd6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (γ2) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (β) and leaky-late (γ1) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.

scholarly journals The UL25 Gene Product of Herpes Simplex Virus Type 1 Is Involved in Uncoating of the Viral Genome

2008 ◽  
Vol 82 (13) ◽  
pp. 6654-6666 ◽  
Author(s):  
Valerie G. Preston ◽  
Jill Murray ◽  
Christopher M. Preston ◽  
Iris M. McDougall ◽  
Nigel D. Stow
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Viral Genome ◽  
Dna Packaging ◽  
Nonpermissive Temperature ◽  
Viral Dna ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT Studies on the herpes simplex virus type 1 UL25-null mutant KUL25NS have shown that the capsid-associated UL25 protein is required at a late stage in the encapsidation of viral DNA. Our previous work on UL25 with the UL25 temperature-sensitive (ts) mutant ts1204 also implicated UL25 in a role at very early times in the virus growth cycle, possibly at the stage of penetration of the host cell. We have reexamined this mutant and discovered that it had an additional ts mutation elsewhere in the genome. The ts1204 UL25 mutation was transferred into wild-type (wt) virus DNA, and the UL25 mutant ts1249 was isolated and characterized to clarify the function of UL25 at the initial stages of virus infection. Indirect immunofluorescence assays and in situ hybridization analysis of virus-infected cells revealed that the mutant ts1249 was not impaired in penetration of the host cell but had an uncoating defect at the nonpermissive temperature. When ts1249-infected cells were incubated initially at the permissive temperature to allow uncoating of the viral genome and subsequently transferred to the restrictive temperature, a DNA-packaging defect was evident. The results suggested that ts1249, like KUL25NS, had a block at a late stage of DNA packaging and that the packaged genome was shorter than the full-length genome. Examination of ts1249 capsids produced at the nonpermissive temperature revealed that, in comparison with wt capsids, they contained reduced amounts of UL25 protein, thereby providing a possible explanation for the failure of ts1249 to package full-length viral DNA.

scholarly journals Determination of Minimum Herpes Simplex Virus Type 1 Components Necessary To Localize Transcriptionally Active DNA to ND10

2003 ◽  
Vol 77 (10) ◽  
pp. 5821-5828 ◽  
Author(s):  
Qiyi Tang ◽  
Luge Li ◽  
Alexander M. Ishov ◽  
Valerie Revol ◽  
Alberto L. Epstein ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Host Protein ◽  
Transcript Accumulation ◽  
Viral Dna ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT DNA viruses such as herpes simplex virus type 1 (HSV-1) appear to start their replicative processes at specific nuclear domains known as ND10. In analyses to determine the minimum viral components needed for transcript accumulation at ND10, we find that a specific viral DNA sequence, OriS, and the viral immediate-early proteins ICP4 and ICP27 are sufficient for a reporter gene placed in cis to the OriS sequence to transcribe at ND10. A chromatin immunoprecipitation assay demonstrated expected critical intermediates in retaining the minimal genome at ND10 for the HSV-1 replication origin through direct or indirect binding to the host protein Daxx. Coimmunoprecipitation assays with antibodies to Daxx and ICP4, ICP27, and ICP8 showed that the respective proteins interact, possibly forming a complex. A potential complex between the origin, early viral DNA-binding protein ICP8 and Daxx did not result in transcription at ND10. Thus, the deposition of transcriptionally active HSV-1 genomes at ND10 is most likely a consequence of retention at ND10 through the interaction of viral genome-bound ICP4 and ICP27 with Daxx. Such a complex might be more likely immobilized at the outside of ND10 by the PML-interacting Daxx than at other nuclear sites.

scholarly journals Hematogenous Vertical Transmission of Herpes Simplex Virus Type 1 in Mice

2006 ◽  
Vol 80 (6) ◽  
pp. 2823-2831 ◽  
Author(s):  
Javier S. Burgos ◽  
Carlos Ramirez ◽  
Fernando Guzman-Sanchez ◽  
Juan M. Alfaro ◽  
Isabel Sastre ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Vertical Transmission ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Severe Disease ◽  
Viral Dna ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that causes severe disease and death in newborn humans but, to date, it remains unclear how neonatal infection occurs. We show here that the vertical transmission of HSV-1 in mice is mainly hematogenous and involves the colonization of the neonate central nervous system (CNS). HSV-1 DNA was mainly detected in the blood and CNS of the offspring born to latently infected mothers; no significant differences were seen between the viral DNA concentrations in the blood of these mothers and their female progeny (either neonate or adult). The administration of acyclovir during gestation reduced or eliminated both the maternal and the neonatal viral DNA in the blood. Embryo transfer was performed to ensure (as far as possible) that only vertical hematogenous infection took place. Immunohistochemical analysis detected viral proteins in the encephalon of the offspring. Immunofluorescence studies provided immunoreactive evidence of HSV-1 proteins in the neurons of the hippocampus and showed that these viruses can molecularly reactivate after hyperthermia. Neonatal HSV-1 infection therefore appears to be mainly caused by hematogenous vertical transmission, and the viruses that colonize the offspring CNS are capable of molecular reactivation after a period of latency.

scholarly journals Functional characterization of the essential tail anchor of the herpes simplex virus type 1 nuclear egress protein pUL34

2011 ◽  
Vol 92 (12) ◽  
pp. 2734-2745 ◽  
Author(s):  
Melanie Ott ◽  
Georg Tascher ◽  
Sarah Haßdenteufel ◽  
Richard Zimmermann ◽  
Jürgen Haas ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Nuclear Egress ◽  
Simplex Virus ◽  
Ta Proteins ◽  
Host Nucleus ◽  
Hsv 1

Release of herpes simplex virus type 1 (HSV-1) nucleocapsids from the host nucleus relies on the nuclear egress complex consisting of the two essential proteins pUL34 and pUL31. The cytoplasmically exposed N-terminal region of pUL34 interacts with pUL31, while a hydrophobic region followed by a short luminal part mediates membrane association. Based on its domain organization, pUL34 was postulated to be a tail-anchor (TA) protein. We performed a coupled in vitro transcription/translation assay to show that membrane insertion of pUL34 occurs post-translationally. Transient transfection and localization experiments in mammalian cells were combined with HSV-1 bacterial artificial chromosome mutagenesis to reveal the functional properties of the essential pUL34 TA. Our data show that a minimal tail length of 15 residues is sufficient for nuclear envelope targeting and pUL34 function. Permutations of the pUL34 TA with orthologous regions of human cytomegalovirus pUL50 or Epstein–Barr virus pBFRF1 as well as the heterologous HSV-1 TA proteins pUL56 or pUS9 or the cellular TA proteins Bcl-2 and Vamp2 revealed that nuclear egress tolerates TAs varying in sequence and hydrophobicity, while a non-α-helical membrane anchor failed to complement the pUL34 function. In conclusion, this study provides the first mechanistic insights into the particular role of the TA of pUL34 in membrane curving and capsid egress from the host nucleus.

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

1998 ◽  
Vol 72 (3) ◽  
pp. 2463-2473 ◽  
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.

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

1996 ◽  
Vol 7 (2) ◽  
pp. 79-85 ◽  
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.

scholarly journals Reconstitution of Herpes Simplex Virus Type 1 Nuclear Capsid Egress In Vitro

2006 ◽  
Vol 80 (19) ◽  
pp. 9741-9753 ◽  
Author(s):  
Gaudeline Rémillard-Labrosse ◽  
Ginette Guay ◽  
Roger Lippé
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Nuclear Membranes ◽  
Nuclear Egress ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Newly assembled herpesvirus capsids travel from the nucleus to the plasma membrane by a mechanism that is poorly understood. Furthermore, the contribution of cellular proteins to this egress has yet to be clarified. To address these issues, an in vitro nuclear egress assay that reproduces the exit of herpes simplex virus type 1 (HSV-1) capsids from nuclei isolated from infected cells was established. As expected, the assay has all the hallmarks of intracellular transport assays, namely, a dependence on time, energy, and temperature. Surprisingly, it is also dependent on cytosol and was slightly enhanced by infected cytosol, suggesting an implication of both host and viral proteins in the process. The capsids escaped these nuclei by budding through the inner nuclear membrane, accumulated as enveloped capsids between the two nuclear membranes, and were released in cytosol exclusively as naked capsids, exactly as in intact cells. This is most consistent with the view that the virus escapes by crossing the two nuclear membranes rather than through nuclear pores. Unexpectedly, nuclei isolated at the nonpermissive temperature from cells infected with a UL26 thermosensitive protease mutant (V701) supported capsid egress. Although electron microscopy, biochemical, and PCR analyses hinted at a likely reconstitution of capsid maturation, DNA encapsidation could not be confirmed by a traditional SQ test. This assay should prove very useful for identification of the molecular players involved in HSV-1 nuclear egress.

Sign in / Sign up

Export Citation Format

Share Document