scholarly journals Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method.

1979 ◽  
Vol 27 (11) ◽  
pp. 1455-1461 ◽  
Author(s):  
B L Hansen ◽  
G N Hansen ◽  
B F Vestergaard
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Reaction Products ◽  
Viral Antigens ◽  
Infected Cells ◽  
Viral Morphogenesis ◽  
Simplex Virus ◽  
Unlabeled Antibody ◽  
Hsv 1

Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.

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 The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge

1998 ◽  
Vol 72 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Tao Peng ◽  
Manuel Ponce-de-Leon ◽  
Hongbin Jiang ◽  
Gary Dubin ◽  
John M. Lubinski ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Antigenic Structure ◽  
Virus Infectivity ◽  
Neutralizing Activity ◽  
Virus Challenge ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.

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 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

2005 ◽  
Vol 79 (8) ◽  
pp. 4730-4743 ◽  
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.

scholarly journals Resistance of mRNA Translation to Acute Endoplasmic Reticulum Stress-Inducing Agents in Herpes Simplex Virus Type 1-Infected Cells Requires Multiple Virus-Encoded Functions

2006 ◽  
Vol 80 (15) ◽  
pp. 7354-7363 ◽  
Author(s):  
Matthew Mulvey ◽  
Carolina Arias ◽  
Ian Mohr
Keyword(s):  
Endoplasmic Reticulum ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Er Stress ◽  
Eif2α Phosphorylation ◽  
Cellular Translation ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Via careful control of multiple kinases that inactivate the critical translation initiation factor eIF2 by phosphorylation of its alpha subunit, the cellular translation machinery can rapidly respond to a spectrum of environmental stresses, including viral infection. Indeed, virus replication produces a battery of stresses, such as endoplasmic reticulum (ER) stress resulting from misfolded proteins accumulating within the lumen of this organelle, which could potentially result in eIF2α phosphorylation and inhibit translation. While cellular translation is exquisitely sensitive to ER stress-inducing agents, protein synthesis in herpes simplex virus type 1 (HSV-1)-infected cells is notably resistant. Sustained translation in HSV-1-infected cells exposed to acute ER stress does not involve the interferon-induced, double-stranded RNA-responsive eIF2α kinase PKR, and it does not require either the PKR inhibitor encoded by the Us11 gene or the eIF2α phosphatase component specified by the γ134.5 gene, the two viral functions known to regulate eIF2α phosphorylation. In addition, although ER stress potently induced the GADD34 cellular eIF2α phosphatase subunit in uninfected cells, it did not accumulate to detectable levels in HSV-1-infected cells under identical exposure conditions. Significantly, resistance of translation to the acute ER stress observed in infected cells requires HSV-1 gene expression. Whereas blocking entry into the true late phase of the viral developmental program does not abrogate ER stress-resistant translation, the presence of viral immediate-early proteins is sufficient to establish a state permissive of continued polypeptide synthesis in the presence of ER stress-inducing agents. Thus, one or more previously uncharacterized viral functions exist to counteract the accumulation of phosphorylated eIF2α in response to ER stress in HSV-1-infected cells.

scholarly journals Herpes Simplex Virus Type 1 Immediate-Early Protein ICP22 Is Required for VICE Domain Formation during Productive Viral Infection

2009 ◽  
Vol 84 (5) ◽  
pp. 2384-2394 ◽  
Author(s):  
Thomas W. Bastian ◽  
Christine M. Livingston ◽  
Sandra K. Weller ◽  
Stephen A. Rice
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Productive Infection ◽  
Domain Formation ◽  
Transfected Cells ◽  
Early Protein ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT During productive infection, herpes simplex virus type 1 (HSV-1) induces the formation of discrete nuclear foci containing cellular chaperone proteins, proteasomal components, and ubiquitinated proteins. These structures are known as VICE domains and are hypothesized to play an important role in protein turnover and nuclear remodeling in HSV-1-infected cells. Here we show that VICE domain formation in Vero and other cells requires the HSV-1 immediate-early protein ICP22. Since ICP22 null mutants replicate efficiently in Vero cells despite being unable to induce VICE domain formation, it can be concluded that VICE domain formation is not essential for HSV-1 productive infection. However, our findings do not exclude the possibility that VICE domain formation is required for viral replication in cells that are nonpermissive for ICP22 mutants. Our studies also show that ICP22 itself localizes to VICE domains, suggesting that it could play a role in forming these structures. Consistent with this, we found that ICP22 expression in transfected cells is sufficient to reorganize the VICE domain component Hsc70 into nuclear inclusion bodies that resemble VICE domains. An N-terminal segment of ICP22, corresponding to residues 1 to 146, is critical for VICE domain formation in infected cells and Hsc70 reorganization in transfected cells. We previously found that this portion of the protein is dispensable for ICP22's effects on RNA polymerase II phosphorylation. Thus, ICP22 mediates two distinct regulatory activities that both modify important components of the host cell nucleus.

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

2002 ◽  
Vol 76 (10) ◽  
pp. 4785-4791 ◽  
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.

scholarly journals The effects of antiviral therapy on the distribution of herpes simplex virus type 1 to ganglionic neurons and its consequences during, immediately following and several months after treatment

2000 ◽  
Vol 81 (10) ◽  
pp. 2385-2396 ◽  
Author(s):  
Alana M. Thackray ◽  
Hugh J. Field
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Reporter Gene ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Continuous Treatment ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

Both famciclovir (FCV) and valaciclovir (VACV) are potent inhibitors of herpes simplex virus type 1 (HSV-1) in a murine cutaneous infection model. The object of the present study was to determine whether either drug had an effect on the anatomical distribution of infected neurons in the peripheral nervous system and to assess the consequences for infected cells during, immediately following and several months after a 9 day period of continuous treatment. Mice were inoculated via the neck with a recombinant strain of HSV-1 expressing the lacZ reporter gene under the immediate-early gene promoter. Sensory ganglia were sampled daily up to day 11 post-inoculation (p.i.) and infected cells were detected by means of the reporter gene product. Ganglia were also removed at 1·5 and 10 months p.i. and latency was assessed by explant co-cultivation and by using in situ hybridization to detect LAT-expressing neurons. While both drugs reduced the severity of acute infection markedly, neither compound completely prevented the relentless distribution of infection among peripheral nervous tissue. Furthermore, there was a difference between the compounds regarding the expression of the reporter gene during and after termination of treatment and in the number of residual LAT-positive neurons.

scholarly journals Phosphorylation of Transcription Factor Sp1 during Herpes Simplex Virus Type 1 Infection

2002 ◽  
Vol 76 (13) ◽  
pp. 6473-6479 ◽  
Author(s):  
Dool-Bboon Kim ◽  
Neal A. DeLuca
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Productive Infection ◽  
E Gene ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The expression of most herpes simplex virus type 1 (HSV-1) immediate-early (IE) and early (E) genes decreases late in productive infection. IE and E promoters contain various binding sites for cellular activators, including sites for Sp1, upstream of the TATA box, while late gene promoters generally lack such sites. To address the possibility that Sp1 function may be altered during the course of infection, the modification state and activity of Sp1 were investigated as a function of infection. Sp1 was quantitatively phosphorylated in HSV-1-infected cells without a significant change in abundance. The kinetics of accumulation of phosphorylated Sp1 immediately preceded the decline in E gene (thymidine kinase gene [tk]) mRNA abundance. Phosphorylation of Sp1 required ICP4; however, the proportion of phosphorylated Sp1 was reduced during infection in the presence of phosphonoacetic acid or in the absence of ICP27. While the DNA binding activity of Sp1 was not greatly affected by phosphorylation, the ability of phosphorylated Sp1 isolated from HSV-infected cells to activate transcription in vitro was decreased. These studies suggest that modification of Sp1 may contribute to the decrease of IE and E gene expression late in infection.

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