Characterization of a neurovirulent aciclovir-resistant variant of herpes simplex virus

Grey, Finn; Sowa, Mike; Collins, Peter; Fenton, Robert J.; Harris, Wendy J.; Snowden, Wendy; Efstathiou, Stacey; Darby, G.

doi:10.1099/vir.0.18881-0

Cited by 25 publications

(35 citation statements)

References 28 publications

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“…In this study, we examined the effects of three tk frameshift mutations found in clinical ACV r isolates on TK expression in HSV-infected cells using plaque autoradiography and a highly sensitive IP-Western blotting technique. For one mutation, G9, our results confirm previous findings (12,16) that reversion can account for much of the TK activity of these viruses (although it is likely that frameshifting also contributes to this [13,16,17]) and extend those findings by detecting and quantifying full-length TK polypeptide synthesis from the mutant with this mutation. For a second mutation, G6, we found that the mutation abolished detectable TK enzyme activity in a plaque autoradiography assay and reduced full-length TK expression ϳ10,000-fold.…”

Section: Discussionsupporting

confidence: 80%

“…In all experiments, fulllength TK levels for G8-FLAG and G9-FLAG were in the range of 0.08 to 0.15% and 0.3 to 1%, respectively. The latter range of values is consistent with amounts of TK enzyme activity measured in lysates of cells infected with a different G9 mutant (12).…”

Section: Levels Of Full-length Tk Polypeptides In G-string Mutant-infsupporting

confidence: 73%

“…Reversion can also account for some of the ability of G8 mutants to reactivate from latency (14,25,26). Nevertheless, there is considerable evidence that much of the TK activity of G8 mutants is a result of ribosomal frameshifting (12,16,20). Plaque autoradiography provides no evidence for reversion of G8 mutants in cell culture (14,17).…”

mentioning

confidence: 99%

“…In the case of G9 mutants, plaque autoradiography studies show that much of their TK activity and ability to reactivate from latency result from reversion to a TK-positive phenotype, often by addition of a 10th G (12,16). Reversion can also account for some of the ability of G8 mutants to reactivate from latency (14,25,26).…”

mentioning

confidence: 99%

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Quantification and Analysis of Thymidine Kinase Expression from Acyclovir-Resistant G-String Insertion and Deletion Mutants in Herpes Simplex Virus-Infected Cells

Pan

Coen

2012

J Virol

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To be clinically relevant, drug-resistant mutants must both evade drug action and retain pathogenicity. Many acyclovir-resistant herpes simplex virus mutants from clinical isolates have one or two base insertions (G8 and G9) or one base deletion (G6) in a homopolymeric run of seven guanines (G string) in the gene encoding thymidine kinase (TK). Nevertheless, G8 and G9 mutants express detectable TK activity and can reactivate from latency in mice, a pathogenicity marker. On the basis of studies using cellfree systems, ribosomal frameshifting can explain this ability to express TK. To investigate frameshifting in infected cells, we constructed viruses that express epitope-tagged versions of wild-type and mutant TKs. We measured TK activity by plaque autoradiography and expression of frameshifted and unframeshifted TK polypeptides using a very sensitive immunoprecipitationWestern blotting method. The G6 mutant expressed ϳ0.01% of wild-type levels of TK polypeptide. For the G9 mutant, consistent with previous results, much TK expression could be ascribed to reversion. For the G8 mutant, from these assays and pulselabeling studies, we determined the ratio of synthesis of frameshifted to unframeshifted polypeptides to be 1:100. The effects of stop codons before or after the G string argue that frameshifting can initiate within the first six guanines. However, frameshifting efficiency was altered by stop codons downstream of the string in the 0 frame. The G8 mutant expressed only 0.1% of the wild-type level of full-length TK, considerably lower than estimated previously. Thus, remarkably low levels of TK are sufficient for reactivation from latency in mice.

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Section: Discussionsupporting

confidence: 80%

Section: Levels Of Full-length Tk Polypeptides In G-string Mutant-infsupporting

confidence: 73%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Quantification and Analysis of Thymidine Kinase Expression from Acyclovir-Resistant G-String Insertion and Deletion Mutants in Herpes Simplex Virus-Infected Cells

Pan

Coen

2012

J Virol

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“…Regarding the restoration of the WT UL41 sequence, homopolymeric tracts of nucleotides such as that found within UL41 are known to be mutational hot spots. Two well-known examples of HSV open reading frames with HFMs that have functional consequences are UL23, which encodes thymidine kinase (52)(53)(54)(55), and Us4, which encodes glycoprotein G (56, 57). Szpara and colleagues have recently identified many other examples of HFMs within the HSV genome in a comparative survey of the complete genomes of 26 different HSV-1 isolates (58).…”

Section: Discussionmentioning

confidence: 99%

The Herpes Simplex Virus 2 Virion-Associated Ribonuclease vhs Interferes with Stress Granule Formation

Finnen

Hay

Dauber

et al. 2014

J Virol

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In a previous study, it was observed that cells infected with herpes simplex virus 2 (HSV-2) failed to accumulate stress granules (SGs) in response to oxidative stress induced by arsenite treatment. As a follow-up to this observation, we demonstrate here that disruption of arsenite-induced SG formation by HSV-2 is mediated by a virion component. Through studies on SG formation in cells infected with HSV-2 strains carrying defective forms of UL41, the gene that encodes vhs, we identify vhs as a virion component required for this disruption. Cells infected with HSV-2 strains producing defective forms of vhs form SGs spontaneously late in infection. In addition to core SG components, these spontaneous SGs contain the viral immediate early protein ICP27 as well as the viral serine/threonine kinase Us3. As part of these studies, we reexamined the frameshift mutation known to reside within the UL41 gene of HSV-2 strain HG52. We demonstrate that this mutation is unstable and can rapidly revert to restore wild-type UL41 following low-multiplicity passaging. Identification of the involvement of virion-associated vhs in the disruption of SG formation will enable mechanistic studies on how HSV-2 is able to counteract antiviral stress responses early in infection. In addition, the ability of Us3 to localize to stress granules may indicate novel roles for this viral kinase in the regulation of translation. IMPORTANCEEukaryotic cells respond to stress by rapidly shutting down protein synthesis and storing mRNAs in cytoplasmic stress granules (SGs). Stoppages in protein synthesis are problematic for all viruses as they rely on host cell machinery to synthesize viral proteins. Thus, many viruses target SGs for disruption or modification. Infection by herpes simplex virus 2 (HSV-2) was previously observed to disrupt SG formation induced by oxidative stress. In this follow-up study, we identify virion host shutoff protein (vhs) as a viral protein involved in this disruption. The identification of a specific viral protein involved in disrupting SG formation is a key step toward understanding how HSV-2 interacts with these antiviral structures. Additionally, this understanding may provide insights into the biology of SGs that may find application in studies on human motor neuron degenerative diseases, like amyotrophic lateral sclerosis (ALS), which may arise as a result of dysregulation of SG formation.

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Antiviral Chemotherapy

Field

Whitley

2010

Topley &Amp; Wilson's Microbiology and Microbial Infections

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Characterization of a neurovirulent aciclovir-resistant variant of herpes simplex virus

Cited by 25 publications

References 28 publications

Quantification and Analysis of Thymidine Kinase Expression from Acyclovir-Resistant G-String Insertion and Deletion Mutants in Herpes Simplex Virus-Infected Cells

Quantification and Analysis of Thymidine Kinase Expression from Acyclovir-Resistant G-String Insertion and Deletion Mutants in Herpes Simplex Virus-Infected Cells

The Herpes Simplex Virus 2 Virion-Associated Ribonuclease vhs Interferes with Stress Granule Formation

Antiviral Chemotherapy

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