The U _L 41 protein of herpes simplex virus 1 degrades RNA by endonucleolytic cleavage in absence of other cellular or viral proteins

Abstract

The herpes simplex virus 1 ORF UL41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or ␤-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3 domain disappears rapidly, a large 5 domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: (i) a GST-vhs protein produced in Escherichia coli, solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST-vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. (ii) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3 untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. (iii) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.endoribonuclease ͉ mRNA ͉ virion host shutoff O ne of the key early events in the replication of herpes simplex virus 1 (HSV-1) is the shutoff of host macromolecular metabolism (1). The shutoff of protein synthesis was mapped to a gene designated U L 41, and the protein product was designated virion host shutoff or vhs (2, 3). During productive infection, copies of vhs protein, which enter the cell as components of the virion tegument, cause the degradation of preexisting and newly transcribed mRNAs during the first few hours after infection (2-4). After the onset of viral transcription, vhs also accelerates the turnover of viral mRNAs, facilitating the sequential expression of different classes of viral genes (3, 5, 6). At later times after infection, newly made vhs is sequestered and rendered inactive by another viral protein, VP16 also known as ␣-trans inducing factor, the product of U L 48 (7). The vast literature of the past decade, reviewed in ref. 8, speaks eloquently of the importance of vhs in the biology of HSV. Some of the key studies, relevant to this report, are as follows: (i) vhs degrades mRNA in the absence of other viral proteins, as shown by inhibition of reporter gene expression in mammalian cells transiently cotransfected with a vhs expression vector (9, 10). (ii) vhs, along with homologues from other alphaherpesviruses, shares amino acid sequence similarity with a larger family of human, yeast, bacterial and phage nucleases and mutations of highly conserved residues, known to be essential for the cat...

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

“…In this series of experiments, HEK 293T cells were transfected with plasmids encoding full-length U L 47 tagged with Myc at the N-terminal (U L 47-N-Myc) and/or full-length ICP27. The cells were harvested 48 h after transfection, and total cell lysates were incubated with GST alone or GST fused to full-length VHS (GST-VHS) as previously reported (30). The electrophoretically separated precipitates were reacted with antibody to the Myc tag.…”

“…The rabbit polyclonal antibody against U L 41 has been described elsewhere (30). Rabbit polyclonal antibody against U L 47 was kindly provided by D. M. Meredith (Leeds University, Leeds, United Kingdom).…”

“…The full length of U L 47 tagged with Myc at the 5′ end and ICP27 ORF was subcloned into pcDNA 3.1(+) transfer vector from previously generated constructs (30). The resulting plasmids were named pU L 47 and pICP27, respectively.…”

The virion-packaged endoribonuclease of herpes simplex virus 1 cleaves mRNA in polyribosomes

“…The first is whether VHS-mRNA effectively degrades RNA that is not bound to eIF4F and ribosomes. Purified VHS-RNase is active on synthetic RNAs, only in the presence of relatively high concentrations of the enzyme (1,2). In the context of infected cells, VHS-mRNA was reported to be inefficient in degrading mRNA in cells lacking eIF4H (25).…”

“…he U L 41 gene of herpes simplex virus 1 (HSV-1) encodes an endoribonuclease with substrate specificity of RNase A and designated the virion host shutoff (VHS)-RNase (1,2). It is packaged in virions and introduced into cells during virus entry.…”