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.

Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides

1994 ◽  
Vol 14 (5) ◽  
pp. 3484-3493
Author(s):  
T J Wu ◽  
G Monokian ◽  
D F Mark ◽  
C R Wobbe
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Full Length ◽  
Early Gene ◽  
Immediate Early ◽  
Simplex Virus

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

scholarly journals Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides.

1994 ◽  
Vol 14 (5) ◽  
pp. 3484-3493 ◽  
Author(s):  
T J Wu ◽  
G Monokian ◽  
D F Mark ◽  
C R Wobbe
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transcriptional Activation ◽  
Deletion Mutant ◽  
Full Length ◽  
Early Gene ◽  
Immediate Early ◽  
Simplex Virus

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

scholarly journals Deletion of the Virion Host Shutoff Protein (vhs) from Herpes Simplex Virus (HSV) Relieves the Viral Block to Dendritic Cell Activation: Potential of vhs− HSV Vectors for Dendritic Cell-Mediated Immunotherapy

2003 ◽  
Vol 77 (6) ◽  
pp. 3768-3776 ◽  
Author(s):  
Laila Samady ◽  
Emanuela Costigliola ◽  
Luci MacCormac ◽  
Yvonne McGrath ◽  
Steve Cleverley ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dendritic Cell ◽  
Cell Activation ◽  
Dendritic Cell Activation ◽  
Virus Growth ◽  
Virion Host Shutoff ◽  
Virion Host Shutoff Protein ◽  
Host Shutoff ◽  
Simplex Virus

ABSTRACT Herpes simplex virus (HSV) infects dendritic cells (DC) efficiently but with minimal replication. HSV, therefore, appears to have evolved the ability to enter DC even though they are nonpermissive for virus growth. This provides a potential utility for HSV in delivering genes to DC for vaccination purposes and also suggests that the life cycle of HSV usually includes the infection of DC. However, DC infected with HSV usually lose the ability to become activated following infection (M. Salio, M. Cella, M. Suter, and A. Lanzavecchia, Eur. J. Immunol. 29:3245-3253, 1999; M. Kruse, O. Rosorius, F. Kratzer, G. Stelz, C. Kuhnt, G. Schuler, J. Hauber, and A. Steinkasserer, J. Virol. 74:7127-7136, 2000). We report that for DC to retain the ability to become activated following HSV infection, the virion host shutoff protein (vhs) must be deleted. vhs usually functions to destabilize mRNA in favor of the production of HSV proteins in permissive cells. We have found that it also plays a key role in the inactivation of DC and is therefore likely to be important for immune evasion by the virus. Here, vhs would be anticipated to prevent DC activation in the early stages of infection of an individual with HSV, reducing the induction of cellular immune responses and thus preventing virus clearance during repeated cycles of virus latency and reactivation. Based on this information, replication-incompetent HSV vectors with vhs deleted which allow activation of DC and the induction of specific T-cell responses to delivered antigens have been constructed. These responses are greater than if DC are loaded with antigen by incubation with recombinant protein.

scholarly journals RNA Degradation Induced by the Herpes Simplex Virus vhs Protein Proceeds 5′ to 3′ In Vitro

2004 ◽  
Vol 78 (23) ◽  
pp. 13391-13394 ◽  
Author(s):  
Jorge Perez-Parada ◽  
Holly A. Saffran ◽  
James R. Smiley
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Degradation ◽  
Host Protein ◽  
Vitro Assay ◽  
Virion Host Shutoff ◽  
Virion Host Shutoff Protein ◽  
Reticulocyte Lysate ◽  
Simplex Virus

ABSTRACT The herpes simplex virus virion host shutoff protein (vhs) is an mRNA-specific RNase that contributes to shutoff of host protein synthesis. We show here that vhs-induced mRNA decay proceeds 5′ to 3′ in an in vitro assay system derived from rabbit reticulocyte lysate.

scholarly journals Herpes Simplex Virus Virion Host Shutoff Protein Requires a Mammalian Factor for Efficient In Vitro Endoribonuclease Activity

2001 ◽  
Vol 75 (3) ◽  
pp. 1172-1185 ◽  
Author(s):  
Patricia Lu ◽  
Frank E. Jones ◽  
Holly A. Saffran ◽  
James R. Smiley
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
In Vitro Translation ◽  
Mrna Turnover ◽  
Virion Host Shutoff ◽  
Virion Host Shutoff Protein ◽  
Host Shutoff ◽  
Simplex Virus ◽  
Endoribonuclease Activity

ABSTRACT The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.

scholarly journals Immediate-Early Expression of the Herpes Simplex Virus Type 1 ICP27 Transcript Is Not Critical for Efficient Replication In Vitro or In Vivo

2004 ◽  
Vol 78 (19) ◽  
pp. 10470-10478 ◽  
Author(s):  
Aixu Sun ◽  
G. V. Devi-Rao ◽  
M. K. Rice ◽  
L. W. Gary ◽  
D. C. Bloom ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Immediate Early ◽  
Wild Type ◽  
Early Expression ◽  
Simplex Virus

ABSTRACT We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.

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