Herpes Simplex Virus Growth, Preparation, and Assay

2014 ◽  
pp. 19-29 ◽  
Author(s):  
Peggy Marconi ◽  
Roberto Manservigi
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Growth ◽  
Simplex Virus

scholarly journals A Single Amino Acid Substitution in Herpes Simplex Virus Type 1 VP16 Inhibits Binding to the Virion Host Shutoff Protein and Is Incompatible with Virus Growth

2003 ◽  
Vol 77 (5) ◽  
pp. 2892-2902 ◽  
Author(s):  
J. Knez ◽  
P. T. Bilan ◽  
J. P. Capone
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Immediate Early ◽  
Virus Growth ◽  
Amino Terminal ◽  
Virion Host Shutoff ◽  
Virion Host Shutoff Protein ◽  
Simplex Virus

ABSTRACT In addition to its well-established role in the activation of herpes simplex virus immediate-early gene transcription, VP16 interacts with and downregulates the function of the virion host shutoff protein (vhs), thereby attenuating vhs-mediated destruction of viral mRNAs and translational arrest at late times of infection. We have carried out two-hybrid analysis in vivo and protein-protein interaction assays in vitro to identify determinants in VP16 necessary for interaction with vhs. The minimal amino-terminal subfragment of VP16 capable of binding to vhs encompassed residues 1 to 345. Alteration of a single leucine at position 344 to alanine (L344A) in the context of the amino-terminal fragment of VP16 containing residues 1 to 404 was sufficient to abolish interaction with vhs in vitro and in vivo. Leu344 could be replaced with hydrophobic amino acids (Ile, Phe, Met, or Val) but not by Asn, Lys, or Pro, indicating that hydrophobicity is an important property of binding to vhs. VP16 harboring a loss-of-function mutation at L344 was not compromised in its ability to interact with host cell factor (HCF-1) or to activate transcription of viral immediate-early genes in transient-transfection assays. Virus complementation assays using the VP16-null virus 8MA and the VP16/vhs double-mutant virus 8MAΔSma showed that VP16(L344A) was able to complement the growth of 8MAΔSma but not 8MA. Thus, a single point mutation in VP16 uncouples binding to vhs from other functions of VP16 required for virus growth and indicates that direct physical association between VP16 and vhs is necessary to sustain a productive infection.

scholarly journals Ras-GAP Binding and Phosphorylation by Herpes Simplex Virus Type 2 RR1 PK (ICP10) and Activation of the Ras/MEK/MAPK Mitogenic Pathway Are Required for Timely Onset of Virus Growth

2000 ◽  
Vol 74 (22) ◽  
pp. 10417-10429 ◽  
Author(s):  
C. C. Smith ◽  
J. Nelson ◽  
L. Aurelian ◽  
M. Gober ◽  
B. B. Goswami
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Virus Growth ◽  
Infected Cells ◽  
Simplex Virus ◽  
Factor C ◽  
In Cells

ABSTRACT We used a herpes simplex virus type 2 (HSV-2) mutant with a deletion in the RR1 (ICP10) PK domain (ICP10ΔPK) and an MEK inhibitor (PD98059) to examine the role of ICP10 PK in virus growth. In HSV-2-infected cells, ICP10 PK binds and phosphorylates the GTPase activating protein Ras-GAP. In vitro binding and peptide competition assays indicated that Ras-GAP N-SH2 and PH domains, respectively, bind ICP10 at phosphothreonines 117 and 141 and a WD40-like motif at positions 160 to 173. Binding and phosphorylation did not occur in cells infected with ICP10ΔPK. GTPase activity was significantly lower in HSV-2- than in ICP10ΔPK-infected cells. Conversely, the levels of activated Ras and mitogen-activated protein kinase (MAPK), and the expression and stabilization of the transcription factor c-Fos were significantly increased in cells infected with HSV-2 or a revertant virus [HSV-2(R)] but not with ICP10ΔPK. PD98059 inhibited MAPK activation and induction-stabilization of c-Fos. Expression from the ICP10 promoter was increased in cells infected with HSV-2 but not with ICP10ΔPK, and increased expression was ablated by PD98059. ICP10 DNA formed a complex with nuclear extracts from HSV-2-infected cells which was supershifted by c-Fos antibody and was not seen with extracts from ICP10ΔPK-infected cells. Complex formation was abrogated by PD98059. Onset of HSV-2 replication was significantly delayed by PD98059 (14 h versus 2 h in untreated cells), a delay similar to that seen for ICP10ΔPK. The data indicate that Ras-GAP phosphorylation by ICP10 PK is involved in the activation of the Ras/MEK/MAPK mitogenic pathway and c-Fos induction and stabilization. This results in increased ICP10 expression and the timely onset of HSV-2 growth.

Transdominant inhibition of herpes simplex virus growth in transgenic mice

Virology ◽  
1992 ◽  
Vol 191 (2) ◽  
pp. 581-588 ◽  
Author(s):  
Colton A. Smith ◽  
Neal Adeluca
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transgenic Mice ◽  
Virus Growth ◽  
Simplex Virus

Herpes Simplex Virus Growth, Preparation, and Assay

2019 ◽  
pp. 57-72 ◽  
Author(s):  
Sereina O. Sutter ◽  
Peggy Marconi ◽  
Anita F. Meier
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Growth ◽  
Simplex Virus

scholarly journals A Protein Encoded by the Herpes Simplex Virus (HSV) Type 1 2-Kilobase Latency-Associated Transcript Is Phosphorylated, Localized to the Nucleus, and Overcomes the Repression of Expression from Exogenous Promoters When Inserted into the Quiescent HSV Genome

2002 ◽  
Vol 76 (8) ◽  
pp. 4056-4067 ◽  
Author(s):  
S. K. Thomas ◽  
C. E. Lilley ◽  
D. S. Latchman ◽  
R. S. Coffin
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Types ◽  
Current Evidence ◽  
Virus Growth ◽  
Exact Function ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus (HSV) is characterized by its ability to establish a latent infection in sensory neurons, from which it can periodically reactivate. The mechanisms of latency, however, remain unclear. The HSV genome is quiescent during latency except for the expression of the latency-associated transcripts (LATs). Although the exact function of the LATs remains obscure, current evidence suggests they are multifunctional and are involved in both establishment of latency and reactivation from latency. The LATs contain several open reading frames (ORFs). One or more of the functions of the LATs could therefore be protein mediated. We have previously reported that deregulated expression of the largest of the HSV type 1 (HSV-1) LAT ORFs (∼274 amino acids) greatly enhances virus growth in cell types that are normally relatively nonpermissive for HSV replication and also that it complements mutations to the immediate-early (IE) gene ICP0 (S. K. Thomas, G. Gough, D. S. Latchman, and R. S. Coffin, J. Virol. 73:6618-6625, 1999). Here we show that LAT ORF expression overcomes the repression of expression from exogenous promoters introduced into the HSV-1 genome which normally occurs in the absence of IE gene expression. To further explore LAT ORF function, we have generated an epitope-tagged LAT ORF, LATmycHis, which forms punctate structures in the infected-cell nucleus reminiscent of the structures formed by ICP0. These are associated with the appearance of a phosphorylated form of the protein and are formed adjacent to, or around the edges of, viral replication compartments. These results provide further evidence that the HSV-1 LAT ORF protein is biologically functional and that the tightly regulated expression of this protein may be important in the wild-type latency phenotype in vivo.

scholarly journals Herpes Simplex Virus Latency-Associated Transcript Encodes a Protein Which Greatly Enhances Virus Growth, Can Compensate for Deficiencies in Immediate-Early Gene Expression, and Is Likely To Function during Reactivation from Virus Latency

1999 ◽  
Vol 73 (8) ◽  
pp. 6618-6625 ◽  
Author(s):  
S. K. Thomas ◽  
G. Gough ◽  
D. S. Latchman ◽  
R. S. ◽  
Coffin
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Early Gene ◽  
Immediate Early ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Virus Growth ◽  
Virus Latency ◽  
Simplex Virus

ABSTRACT Herpes simplex virus types 1 and 2 (HSV1 and HSV2) enter and reactivate from latency in sensory neurons, although the events governing these processes are little understood. During latency, only the latency-associated transcripts (LATs) are produced. However, although the LAT RNAs were described ≈10 years ago, their function remains ambiguous. Mutations affecting the LATs have minimal effects other than a small reduction in establishment of and reactivation from latency in some cases. Mutations in putative LAT-contained open reading frames (ORFs) have so far shown no effect. The LATs consist of a large species from which smaller (≈2 kb), nuclear, nonlinear LATs which are abundant during latency are spliced. Thus, translation of ORFs in these smaller LATs would not usually be expected to be possible, and if expressed at all, their expression might be tightly regulated. Here we show that deregulated expression of the largest HSV1 2-kb LAT-contained ORF in various cells of neuronal and nonneuronal origin greatly enhances virus growth in a manner specific to HSV1—the HSV1 LAT ORF has no effect on the growth of HSV2. Similar results of enhanced growth were found when the HSV1 LAT ORF was constitutively expressed from within the HSV1 genome. The mechanism of LAT ORF action was strongly suggested to be by substituting for deficiencies in immediate-early (IE) gene expression (particularly ICP0), because deregulated LAT ORF expression, as well as enhancing wild-type virus growth, was also found to allow efficient growth of viruses with mutations in ICP0 or VMW65. Such viruses otherwise exhibit considerable growth defects. IE gene expression deficiencies are often the block to productive infection in nonpermissive cells and are also evident during latency. These results, which we show to be protein- rather than RNA-mediated effects, strongly suggest a function of the tightly regulated expression of a LAT ORF-encoded protein in the reactivation from HSV latency.

scholarly journals The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

1998 ◽  
Vol 72 (11) ◽  
pp. 9131-9141 ◽  
Author(s):  
C. C. Smith ◽  
T. Peng ◽  
M. Kulka ◽  
L. Aurelian
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Replication ◽  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Wild Type ◽  
Virus Growth ◽  
Simplex Virus

ABSTRACT The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.

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