The Herpes Simplex Virus vhs Protein Induces Endoribonucleolytic Cleavage of Target RNAs in Cell Extracts

ABSTRACT The herpes simplex virus virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. Previous studies have demonstrated that extracts from HSV-infected cells and partially purified HSV virions display vhs-dependent RNase activity and that vhs is sufficient to trigger accelerated RNA degradation when expressed as the only HSV protein in an in vitro translation system derived from rabbit reticulocytes. We have used the rabbit reticulocyte translation system to characterize the mode of vhs-induced RNA decay in more detail. We report here that vhs-dependent RNA decay proceeds through endoribonucleolytic cleavage, is not affected by the presence of a 5′ cap or a 3′ poly(A) tail in the RNA substrate, requires Mg2+, and occurs in the absence of ribosomes. Intriguingly, sites of preferential initial cleavage were clustered over the 5′ quadrant of one RNA substrate that was characterized in detail. The vhs homologue of pseudorabies virus also induced accelerated RNA decay in this in vitro system.

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Structural Variability of the Herpes Simplex Virus 1 GenomeIn VitroandIn Vivo

Journal of Virology ◽

10.1128/jvi.00223-12 ◽

2012 ◽

Vol 86 (16) ◽

pp. 8592-8601 ◽

Cited By ~ 9

Author(s):

Charlotte Mahiet ◽

Ayla Ergani ◽

Nicolas Huot ◽

Nicolas Alende ◽

Ahmed Azough ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Considerable Proportion ◽

Inverted Repeats ◽

Herpes Simplex Virus 1 ◽

Cell Extracts ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus 1 (HSV-1) is a human pathogen that leads to recurrent facial-oral lesions. Its 152-kb genome is organized in two covalently linked segments, each composed of a unique sequence flanked by inverted repeats. Replication of the HSV-1 genome produces concatemeric molecules in which homologous recombination events occur between the inverted repeats. This mechanism leads to four genome isomers (termed P, IS, IL, and ILS) that differ in the relative orientations of their unique fragments. Molecular combing analysis was performed on DNA extracted from viral particles and BSR, Vero, COS-7, and Neuro-2a cells infected with either strain SC16 or KOS of HSV-1, as well as from tissues of experimentally infected mice. Using fluorescence hybridization, isomers were repeatedly detected and distinguished and were accompanied by a large proportion of noncanonical forms (40%). In both cell and viral-particle extracts, the distributions of the four isomers were statistically equivalent, except for strain KOS grown in Vero and Neuro-2a cells, in which P and IS isomers were significantly overrepresented. In infected cell extracts, concatemeric molecules as long as 10 genome equivalents were detected, among which, strikingly, the isomer distributions were equivalent, suggesting that any such imbalance may occur during encapsidation.In vivo, for strain KOS-infected trigeminal ganglia, an unbalanced distribution distinct from the onein vitrowas observed, along with a considerable proportion of noncanonical assortment.

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Analysis of the Requirement for Glycoprotein M in Herpes Simplex Virus Type 1 Morphogenesis

Journal of Virology ◽

10.1128/jvi.78.2.1039-1041.2004 ◽

2004 ◽

Vol 78 (2) ◽

pp. 1039-1041 ◽

Cited By ~ 30

Author(s):

Helena Browne ◽

Susanne Bell ◽

Tony Minson

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Pseudorabies Virus ◽

Glycoprotein E ◽

Virion Assembly ◽

Simplex Virus

ABSTRACT A mutant of herpes simplex virus type 1 lacking both glycoprotein M and glycoprotein E was marginally compromised in terms of its in vitro growth characteristics. This finding is in marked contrast to a similar mutant of the related alphaherpesvirus, pseudorabies virus (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999), and suggests that the glycoprotein requirements for virion assembly may vary among different members of this family of viruses.

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Assembly of the Herpes Simplex Virus Procapsid from Purified Components and Identification of Small Complexes Containing the Major Capsid and Scaffolding Proteins

Journal of Virology ◽

10.1128/jvi.73.5.4239-4250.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 4239-4250 ◽

Cited By ~ 90

Author(s):

William W. Newcomb ◽

Fred L. Homa ◽

Darrell R. Thomsen ◽

Benes L. Trus ◽

Naiqian Cheng ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Capsid Protein ◽

Major Capsid Protein ◽

Scaffolding Protein ◽

Oligomeric State ◽

Cell Extracts ◽

Simplex Virus ◽

Hsv 1

ABSTRACT An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37°C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C1-VP232) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to ∼30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.

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Characterization of mutants of herpes simplex viruses, types 1 and 2, that produce fragments of the thymidine kinase polypeptide

Canadian Journal of Microbiology ◽

10.1139/m83-063 ◽

1983 ◽

Vol 29 (4) ◽

pp. 385-393

Author(s):

Timothy M. -P. Block ◽

Nancy J. Kuhn ◽

Karen A. Kustas ◽

William A. Held ◽

Kenneth Gross ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Thymidine Kinase ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

In Vitro Translation ◽

Herpes Simplex Viruses ◽

Simplex Virus ◽

Hsv 1

Seven tk− mutants of herpes simplex virus, type 2 (HSV-2), and three tk− mutants of herpes simplex virus, type 1 (HSV-1), were isolated which did not produce the thymidine kinase (TK) polypeptides but formed smaller polypeptides not seen in wild-type infected cells. Positive TK mRNA selection by hybridization to the cloned tk genes followed by in vitro translation identified the TK polypeptides. Comparisons of the products of partial proteolysis of the polypeptides of four HSV-2 and two HSV-1 tk− mutants to those of the parental TK polypeptides indicated that, in each case, the novel polypeptide was a fragment of the TK polypeptide, showing that these mutants have defects in the structural gene for tk. HSV-2 mutants of this sort have not been previously described. They and the HSV-1 mutants are similar to HSV-1 mutants reported previously. In addition, it was found that TK mRNA was present early in infection but was absent late in infection, suggesting that the shutoff of TK synthesis is due to message degradation. Also, HSV-2 TK mRNA did not hybridize to the cloned HSV-1 tk gene indicating that these genes have extensively diverged.

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Expression of herpes simplex virus type 1 DNA polymerase gene by in vitro translation and effects of gene deletions on activity.

Journal of Virology ◽

10.1128/jvi.62.9.3224-3232.1988 ◽

1988 ◽

Vol 62 (9) ◽

pp. 3224-3232 ◽

Cited By ~ 24

Author(s):

D I Dorsky ◽

C S Crumpacker

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Polymerase ◽

Herpes Simplex Virus Type ◽

In Vitro Translation ◽

Polymerase Gene ◽

Gene Deletions ◽

Simplex Virus

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

Journal of Virology ◽

10.1128/jvi.78.23.13391-13394.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13391-13394 ◽

Cited By ~ 16

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.

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

Journal of Virology ◽

10.1128/jvi.75.3.1172-1185.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1172-1185 ◽

Cited By ~ 24

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.

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Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1

Viruses ◽

10.3390/v13020196 ◽

2021 ◽

Vol 13 (2) ◽

pp. 196

Author(s):

Sara Artusi ◽

Emanuela Ruggiero ◽

Matteo Nadai ◽

Beatrice Tosoni ◽

Rosalba Perrone ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Dna Replication ◽

Herpes Simplex ◽

Antiviral Activity ◽

Herpes Simplex Virus 1 ◽

Tem Analysis ◽

Infected Cells ◽

Simplex Virus ◽

Hsv 1

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.

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