Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for accumulation of mRNA

Taddeo, Brunella; Sciortino, Maria Teresa; Zhang, Weiran; Roizman, Bernard

doi:10.1073/pnas.0705245104

Cited by 67 publications

(85 citation statements)

References 29 publications

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“…The prime viral gene product that could be responsible for the decrease in LATs or miRNAs is the viral endoribonuclease encoded by the U L 41 gene. In cell culture, modified forms of LATs accumulate late in infection (18), after the endoribonuclease is neutralized by two viral proteins, VP16 and virion protein 22 (19). Whether the degradation of LATs or miRNAs is caused by endoribonuclease encoded by the U L 41 gene remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs

Zhou

Roizman

2011

Proc. Natl. Acad. Sci. U.S.A.

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In cell cultures, HSV-1 replication is initiated by recruitment by virion protein 16 of transcriptional factors and histone-modifying enzymes to immediate early (α) gene promoters. HSV establishes latent infections characterized by suppression of viral gene expression except for latency-associated transcripts (LATs) and miRNAs. The latent virus reactivates in stressed neurons. A fundamental question is how reactivation initiates in the absence of virion protein 16. We report the following findings in the ganglion explant model. (i) Anti-nerve growth factor antibody accelerated the reactivation of latent virus. Viral mRNAs were detected as early as 9 h after explantation. (ii) After explantation the amounts of viral mRNAs increased whereas amounts of miRNAs and LATs decreased. The decrease in miRNAs and LATs required ongoing protein synthesis, raising the possibility that LAT and miRNAs were degraded by a viral gene product. (iii) The expression of viral genes in explanted ganglia was disordered rather than sequentially ordered as in infected cells in culture. These findings suggest that in reactivating ganglia gene expression is totally derepressed and challenge the current models in that establishment of or exit from latency could not be dependent on the suppression or activation of single or small clusters of viral genes. Finally, miRNAs and LATs reached peak levels 9-11 d after corneal inoculation, thus approximating the pattern of virus replication in these ganglia. These findings suggest that the patterns of accumulation of LATs and miRNAs reflect many different stages in the infection of neurons.T o initiate infection in cell culture and presumably also at the portal of entry into the body, the HSV-1 capsid delivers the DNA to the nucleus. Concurrently, virion protein 16 (VP16), a key viral protein packaged in the virion and delivered to the nucleus, recruits the cellular factors octamer-binding protein 1 (Oct1), host cell factor 1 (HCF1), lysine-specific demethylase 1 (LSD1), circadian locomotor output cycles kaput (CLOCK) histone acetyl transferase, and other transcriptional factors to initiate a cascade of viral gene expression that begins with immediate early (α) genes followed by early (β) and late (γ) genes (1-5). VP16 is encoded by a γ gene expressed late in infection (1). In all, the transcriptional program yields almost 100 different transcripts. In contrast, in latently infected neurons viral gene expression is limited to the latency-associated transcript (LAT) and approximately six or more miRNAs (6, 7). Given the requirement for VP16 to initiate viral replication in productively infected cells, the question arises as to how virus replication initiates in stressed neurons harboring latent virus. Among the many hypotheses, two stand out: That VP16 is expressed first or that in neurons α gene expression can ensue in the absence of VP16.Resolution of this problem requires analyses of the reactivation in ganglia under conditions that are physiologically similar to those that occur in vivo. In the st...

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Section: Discussionmentioning

confidence: 99%

HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs

Zhou

Roizman

2011

Proc. Natl. Acad. Sci. U.S.A.

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110

132

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“…HEK 293T Cell Transient Transfection. The procedures for transfection HEK 293T cells were described previously (14). The cells were harvested 48 h after transfection and lysed according to the protocol for the subsequent GST pull-down assay.…”

Section: Methodsmentioning

confidence: 99%

“…The procedures of pull-down were performed as previously described (14). Briefly, transfected HEK 293T cells were lysed in GST lysis buffer [20 mM Tris (pH 8.0)/1 mM EDTA/1% Nonidet P-40/200 mM NaCl/ 0.1 mM sodium orthovanadate/10 mM NaF/2 mM DTT/protease inhibitor mixture (Complete Protease; Roche Diagnostics] on ice for 1 h. Insoluble material was pelleted by centrifugation at maximum speed in a 5415C centrifuge (Eppendorf) for 10 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…The primary function of VHSRNase appears to be the degradation of mRNAs made in response to infection, but it also prevents, by a mechanism not fully understood, the activation of protein kinase R (10-12). At late stages in the viral replicative cycle, virion host shutoff (VHS)-RNase activity is blocked or neutralized by two late proteins, virion protein (VP) 16 and VP22, encoded by U L 48 and U L 49 ORFs, respectively (13,14). The RNase cleaves mRNA in polyribosomes (15).…”

mentioning

confidence: 99%

“…The Northern blot analysis was done as described previously (14), with minor modifications. Briefly, 8 μg of RNA were loaded onto denaturing formaldehyde gel and probed with random hexanucleotide-primed 32 P-labeled fragment of indicated viral (ICP0, ICP8, and VP16) or cellular (GAPDH, IEX-1) genes upon transfer onto a nylon membrane.…”

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Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the U _L 47 tegument protein

Shu

Taddeo

Zhang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Herpes simplex virus 1 (HSV-1) encodes an endoribonuclease that is responsible for the shutoff of host protein synthesis [virion host shutoff (VHS)-RNase]. The VHS-RNase released into cells during infection targets differentially four classes of mRNAs. Thus, (a) VHSRNase degrades stable cellular mRNAs and α (immediate early) viral mRNAs; (b) it stabilizes host stress response mRNAs after deadenylation and subsequent cleavage near the adenylate-uridylate (AU)-rich elements; (c) it does not effectively degrade viral β or γ mRNAs; and (d) it selectively spares from degradation a small number of cellular mRNAs. Current evidence suggests that several viral and at least one host protein (tristetraprolin) regulate its activity. Thus, virion protein (VP) 16 and VP22 neutralize the RNase activity at late times after infection. By binding to AU-rich elements via its interaction with tristetraprolin, the RNase deadenylates and cleaves the mRNAs in proximity to the AU-rich elements. In this report we show that another virion protein, U L 47, brought into the cell during infection, attenuates the VHS-RNase activity with respect to stable host and viral α mRNAs and effectively blocks the degradation of β and γ mRNAs, but it has no effect on the processing of AU-rich mRNAs. The properties of U L 47 suggest that it, along with the α protein infected cell protein 27, attenuates degradation of mRNAs by the VHS-RNase through interaction with the enzyme in polyribosomes. Mutants lacking both VHS-RNase and U L 47 overexpress α genes and delay the expression of β and γ genes, suggesting that overexpression of α genes inhibits the downstream expression of early and late genes.innate immunity | host response A ttachment and entry of herpes simplex virus 1 (HSV-1) provokes powerful innate immune responses to the virus. HSV-1 blocks the responses in two fundamentally different ways, by bringing into cells at the time of entry both tegument proteins and prepackaged mRNAs and by encoding proteins made immediately after infection with numerous functions designed to block host innate immune responses (1-5). Among the best-known and highly effective tegument proteins actively blocking host responses is the virion host shutoff RNase encoded by the U L 41 ORF (6-8). This RNase is an endoribonuclease with the specificity of RNase A (9), and it is active immediately after entry of the virus into cells and during the initial stages of infection. The primary function of VHSRNase appears to be the degradation of mRNAs made in response to infection, but it also prevents, by a mechanism not fully understood, the activation of protein kinase R (10-12). At late stages in the viral replicative cycle, virion host shutoff (VHS)-RNase activity is blocked or neutralized by two late proteins, virion protein (VP) 16 and VP22, encoded by U L 48 and U L 49 ORFs, respectively (13,14). The RNase cleaves mRNA in polyribosomes (15). Another partner of the VHS-RNase present in polyribosomes is infected cell protein (ICP) 27, an α (immediate early) multifunctional reg...

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Bleach gel: A simple agarose gel for analyzing RNA quality

2012

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RNA-based applications requiring high quality, non-degraded RNA are a foundational element of many research studies. As such, it is paramount that the integrity of experimental RNA is validated prior to cDNA synthesis or other downstream applications. In the absence of expensive equipment such as microfluidic electrophoretic devices, and as an alternative to the costly and time-consuming standard formaldehyde gel, RNA quality can be quickly analyzed by adding small amounts of commercial bleach to TAE buffer-based agarose gels prior to electrophoresis. In the presence of low concentrations of bleach, the secondary structure of RNA is denatured and potential contaminating RNases are destroyed. Because of this, the ‘bleach gel’ is a functional approach that addresses the need for an inexpensive and safe way to evaluate RNA integrity and will improve the ability of researchers to rapidly analyze RNA quality.

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Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for accumulation of mRNA

Cited by 67 publications

References 29 publications

HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs

HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs

Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the U _L 47 tegument protein

Bleach gel: A simple agarose gel for analyzing RNA quality

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Interaction of herpes simplex virus RNase with VP16 and VP22 is required for the accumulation of the protein but not for accumulation of mRNA

Cited by 67 publications

References 29 publications

HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs

HSV-1 gene expression from reactivated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs

Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the U L 47 tegument protein

Bleach gel: A simple agarose gel for analyzing RNA quality

Contact Info

Product

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About

Selective degradation of mRNAs by the HSV host shutoff RNase is regulated by the U _L 47 tegument protein