scholarly journals The N Terminus of the Herpes Simplex Virus Type 1 Triplex Protein, VP19C, Cannot Be Detected on the Surface of the Capsid Shell by Using an Antibody (Hemagglutinin) Epitope Tag

2007 ◽  
Vol 81 (15) ◽  
pp. 8367-8370 ◽  
Author(s):  
Marieta Solé ◽  
Edward M. Perkins ◽  
Augusto Frisancho ◽  
Eugene Huang ◽  
Prashant Desai
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Vero Cells ◽  
Amino Terminus ◽  
Epitope Tag ◽  
Recombinant Viruses ◽  
Capsid Shell ◽  
N Terminus ◽  
Simplex Virus

ABSTRACT The herpes simplex virus (HSV) triplex is a complex of three protein subunits, VP19C and a dimer of VP23 that is essential for capsid assembly. We have derived HSV-1 recombinant viruses that contain monomeric red fluorescent protein (mRFP1), a Flu hemagglutinin (HA) epitope, and a six-histidine tag fused to the amino terminus of VP19C. These viruses were capable of growth on Vero cells, indicating that the amino terminus of VP19C could tolerate these fusions. By use of immunoelectron microscopy methods, capsids that express VP19C-mRFP but not VP19C-HA were labeled with gold particles when incubated with the corresponding antibody. Our conclusion from the data is that a large tag at the N terminus of VP19C was sufficiently exposed on the capsid surface for polyclonal antibody reactivity, while the small HA epitope was inaccessible to the antibody. These data indicate that an epitope tag at the amino terminus of VP19C is not exposed at the capsid surface for reactivity to its antibody.

scholarly journals Glycoprotein K Specified by Herpes Simplex Virus Type 1 Is Expressed on Virions as a Golgi Complex-Dependent Glycosylated Species and Functions in Virion Entry

2001 ◽  
Vol 75 (24) ◽  
pp. 12431-12438 ◽  
Author(s):  
Timothy P. Foster ◽  
Galena V. Rybachuk ◽  
Konstantin G. Kousoulas
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Golgi Complex ◽  
Vero Cells ◽  
Wild Type ◽  
Epitope Tag ◽  
Glycoprotein K ◽  
Domain Iii ◽  
Simplex Virus

ABSTRACT To facilitate detection of glycoprotein K (gK) specified by herpes simplex virus, a 12-amino-acid epitope tag was inserted within gK domain III. Recombinant virus gKprotC-DIII, expressing the tagged gK, was isolated. This virus formed wild-type plaques and replicated as efficiently as the wild-type KOS virus in Vero cells. Anti-protein C MAb detected high-mannose and Golgi complex-dependent glycosylated gK within cells as well as on purified virions. The gK-null virus ΔgK (gK−/−) entered Vero cells substantially more slowly than the wild-type KOS (gK+/+), while ΔgK virus grown in complementing VK302 cells (gK−/+) entered with entry kinetics similar to those of the KOS virus.

scholarly journals The Herpes Simplex Virus Type 1 UL20 Protein Modulates Membrane Fusion Events during Cytoplasmic Virion Morphogenesis and Virus-Induced Cell Fusion

2004 ◽  
Vol 78 (10) ◽  
pp. 5347-5357 ◽  
Author(s):  
Timothy P. Foster ◽  
Jeffrey M. Melancon ◽  
Joel D. Baines ◽  
Konstantin G. Kousoulas
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Vero Cells ◽  
Recombinant Viruses ◽  
Cytoplasmic Vacuoles ◽  
Virion Morphogenesis ◽  
Simplex Virus

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL20 protein is an important determinant for virion morphogenesis and virus-induced cell fusion. A precise deletion of the UL20 gene in the HSV-1 KOS strain was constructed without affecting the adjacent UL20.5 gene. The resultant KOS/UL20-null virus produced small plaques of 8 to 15 cells in Vero cells while it produced wild-type plaques on the complementing cell line G5. Electron microscopic examination of infected cells revealed that the KOS/UL20-null virions predominantly accumulated capsids in the cytoplasm while a small percentage of virions were found as enveloped virions within cytoplasmic vacuoles. Recently, it was shown that UL20 expression was necessary and sufficient for cell surface expression of gK (T. P. Foster, X. Alvarez, and K. G. Kousoulas, J. Virol. 77:499-510, 2003). Therefore, we investigated the effect of UL20 on virus-induced cell fusion caused by syncytial mutations in gB and gK by constructing recombinant viruses containing the gBsyn3 or gKsyn1 mutations in a UL20-null genetic background. Both recombinant viruses failed to cause virus-induced cell fusion in Vero cells while they readily caused fusion of UL20-null complementing G5 cells. Ultrastructural examination of UL20-null viruses carrying the gBsyn3 or gKsyn1 mutation revealed a similar distribution of virions as the KOS/UL20-null virus. However, cytoplasmic vacuoles contained aberrant virions having multiple capsids within a single envelope. These multicapsid virions may have been formed either by fusion of viral envelopes or by the concurrent reenvelopment of multiple capsids. These results suggest that the UL20 protein regulates membrane fusion phenomena involved in virion morphogenesis and virus-induced cell fusion.

scholarly journals ATP-Dependent Localization of the Herpes Simplex Virus Capsid Protein VP26 to Sites of Procapsid Maturation

2000 ◽  
Vol 74 (3) ◽  
pp. 1468-1476 ◽  
Author(s):  
Jung Hee I. Chi ◽  
Duncan W. Wilson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Cold Sensitivity ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Protein Fusion ◽  
Capsid Shell ◽  
Green Fluorescent ◽  
Simplex Virus

ABSTRACT The herpes simplex virus type 1 (HSV-1) capsid shell is composed of four major polypeptides, VP5, VP19c, VP23, and VP26. VP26, a 12-kDa polypeptide, is associated with the tips of the capsid hexons formed by VP5. Mature capsids form upon angularization of the shell of short-lived, fragile spherical precursors termed procapsids. The cold sensitivity and short-lived nature of the procapsid have made its isolation and biochemical analysis difficult, and it remains unclear whether procapsids contain bound VP26 or whether VP26 is recruited following shell angularization. By indirect immunocytochemical analysis of virally expressed VP26 and by direct visualization of a transiently expressed VP26-green fluorescent protein fusion, we show that VP26 fails to specifically localize to intranuclear procapsids accumulated following incubation of the temperature-sensitive HSV mutanttsProt.A under nonpermissive conditions. However, following a downshift to the permissive temperature, which allows procapsid maturation to proceed, VP26 was seen to concentrate at intranuclear sites which also contained epitopes specific to mature, angularized capsids. Like the formation of these epitopes, the association of VP26 with maturing capsids was blocked in a reversible fashion by the depletion of intracellular ATP. We conclude that unlike the other major capsid shell proteins, VP26 is recruited in an ATP-dependent fashion after procapsid maturation begins.

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

2008 ◽  
Vol 83 (4) ◽  
pp. 1660-1668 ◽  
Author(s):  
Anna Maria Copeland ◽  
William W. Newcomb ◽  
Jay C. Brown
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Capsid Protein ◽  
Fluorescent Protein ◽  
Vero Cells ◽  
Tegument Protein ◽  
Nuclear Pore ◽  
Nuclear Surface ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT 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.

scholarly journals Localization of Herpes Simplex Virus Type 1 UL37 in the Golgi Complex Requires UL36 but Not Capsid Structures

2008 ◽  
Vol 82 (22) ◽  
pp. 11354-11361 ◽  
Author(s):  
Prashant Desai ◽  
Gerry L. Sexton ◽  
Eugene Huang ◽  
Stanley Person
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Golgi Complex ◽  
Herpes Simplex Virus Type ◽  
Fluorescent Protein ◽  
Vero Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL37 gene encodes a 120-kDa polypeptide which resides in the tegument structure of the virion and is important for morphogenesis. The goal of this study was to use green fluorescent protein (GFP) to follow the fate of UL37 within cells during the normal course of virus replication. GFP was inserted in frame at the C terminus of UL37 to generate a fluorescent-protein-tagged UL37 polypeptide. A virus designated K37eGFP, which replicated normally on Vero cells, was isolated and was shown to express the fusion polypeptide. When cells infected with this virus were examined by confocal microscopy, the fluorescence was observed to be predominantly cytoplasmic. As the infection progressed, fluorescence began to accumulate in a juxtanuclear structure. Mannosidase II and giantin were observed to colocalize with UL37eGFP at these structures, as judged by immunofluorescence assays. Therefore, UL37 traffics to the Golgi complex during infection. A VP26mRFP marker (red fluorescent protein fused to VP26) was recombined into K37eGFP, and when cells infected with this “dual-color” virus were examined, colocalization of the red (capsid) and green (UL37) fluorescence in the Golgi structure was observed. Null mutations in VP5 (ΔVP5), which abolished capsid assembly, and in UL36 (Δ36) were recombined into the K37eGFP virus genome. In cells infected with K37eGFP/ΔVP5, localization of UL37eGFP to the Golgi complex was similar to that for the parental virus (K37eGFP), indicating that trafficking of UL37eGFP to the Golgi complex did not require capsid structures. Confocal analysis of cells infected with K37eGFP/Δ36 showed that, in the absence of UL36, accumulation of UL37eGFP at the Golgi complex was not evident. This indicates an interaction between these two proteins that is important for localization of UL37 in the Golgi complex and thus possibly for cytoplasmic envelopment of the capsid. This is the first demonstration of a functional role for UL36:UL37 interaction in HSV-1-infected cells.

scholarly journals The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

2010 ◽  
Vol 84 (17) ◽  
pp. 8596-8606 ◽  
Author(s):  
Vladimir N. Chouljenko ◽  
Arun V. Iyer ◽  
Sona Chowdhury ◽  
Joohyun Kim ◽  
Konstantin G. Kousoulas
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Protein Complex ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Glycoprotein K ◽  
Simplex Virus

ABSTRACT Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.

scholarly journals The Polysulfonated Compound Suramin Blocks Adsorption and Lateral Difusion of Herpes Simplex Virus Type-1 in Vero Cells

Virology ◽  
1999 ◽  
Vol 258 (1) ◽  
pp. 141-151 ◽  
Author(s):  
José S. Aguilar ◽  
Marcia Rice ◽  
Edward K. Wagner
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Vero Cells ◽  
Simplex Virus

scholarly journals Herpes Simplex Virus Type 1 Corneal Infection Results in Periocular Disease by Zosteriform Spread

2001 ◽  
Vol 75 (11) ◽  
pp. 5069-5075 ◽  
Author(s):  
Bretton C. Summers ◽  
Todd P. Margolis ◽  
David A. Leib
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Skin Lesions ◽  
Ocular Infection ◽  
Trigeminal Ganglia ◽  
Corneal Infection ◽  
Recombinant Viruses ◽  
Viral Spread ◽  
Reporter Activity ◽  
Simplex Virus

ABSTRACT In humans and animal models of herpes simplex virus infection, zosteriform skin lesions have been described which result from anterograde spread of the virus following invasion of the nervous system. Such routes of viral spread have not been fully examined following corneal infection, and the possible pathologic consequences of such spread are unknown. To investigate this, recombinant viruses expressing reporter genes were generated to quantify and correlate gene expression with replication in eyes, trigeminal ganglia, and periocular tissue. Reporter activity peaked in eyes 24 h postinfection and rapidly fell to background levels by 48 h despite the continued presence of viral titers. Reporter activity rose in the trigeminal ganglia at 60 h and peaked at 72 h, concomitant with the appearance and persistence of infectious virus. Virus was present in the periocular skin from 24 h despite the lack of significant reporter activity until 84 h postinfection. This detection of reporter activity was followed by the onset of periocular disease on day 4. Corneal infection with a thymidine kinase-deleted reporter virus displayed a similar profile of reporter activity and viral titer in the eyes, but little or no detectable activity was observed in trigeminal ganglia or periocular tissue. In addition, no periocular disease symptoms were observed. These findings demonstrate that viral infection of periocular tissue and subsequent disease development occurs by zosteriform spread from the cornea to the periocular tissue via the trigeminal ganglion rather than by direct spread from cornea to the periocular skin. Furthermore, clinical evidence is discussed suggesting that a similar mode of spreading and disease occurs in humans following primary ocular infection.

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.

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