The Nuclear-Cytoplasmic Shuttling of Virion Host Shutoff RNase Is Enabled by pU _L 47 and an Embedded Nuclear Export Signal and Defines the Sites of Degradation of AU-Rich and Stable Cellular mRNAs

Abstract

The herpes simplex virus host shutoff RNase (VHS-RNase) is the major early block of host responses to infection. VHS-RNase is introduced into cells during infection and selectively degrades stable mRNAsT he virion host shutoff (VHS) RNase is a late (␥ 2 ) tegument protein carried into the cell during infection. It plays a key role in blocking host responses to infection capable of curtailing viral replication (1-4). It functions as an endoribonuclease with the specificity of RNase A (5). VHS-RNase binds to eIF4H and cleaves stable mRNAs in polyribosomes 3= to the 5= cap structure. The RNA is then degraded processively 5= to 3= (6). The main target of the VHS-RNase are the short-lived stress response mRNAs characterized by the presence AU-rich elements in their 3= untranslated regions (3= UTR) targeted by tristetraprolin (TTP) (7). The VHS-RNase binds to tristetraprolin at the AU-rich elements and cleaves the RNA 5= to the AU-rich element. The 3= portion of the cleaved RNA is rapidly degraded 5= to 3=. The 5= portion of the cleaved AU-rich mRNA lingers for many hours in the cytoplasm, possibly because of a dearth in enzymes capable of cleaving the RNA 3= to 5= (8). Both the stable and the AU-rich short-lived mRNAs are also deadenylated in this process. It is noteworthy that on transfection, VHS-RNase degrades its own mRNA as well as the mRNAs of cotransfected genes (9).In infected cells, VHS is regulated by several viral proteins. Thus, late in infection, VHS is completely neutralized by two late proteins, VP16 and VP22, prior to packaging in virions (9, 10). Early in infection, the function of VHS is regulated by pU L 47, a tegument protein (11). Recent studies have shown that on entry into cells infected with wild-type virus, VHS-RNase targets mRNAs made before infection and AU-rich mRNAs, but largely spares viral mRNAs made after infection (12). The selective sparing of stable mRNAs made after infection has been shown to be due to the interaction of VHS with pU L 47 (11). pU L 47 has no effect on the degradation of AU-rich mRNAs. It has been reported to shuttle between the nucleus and cytoplasm and to bind poly(A) binding protein (13,14).The fundamental question posed by the present study was to define the subcellular compartment in which the VHS-RNase newly introduced into the infected cell functions. To resolve this question, we examined the coding sequence of VHS for nuclear localization and nuclear export motifs. We did not identify an obvious nuclear localization motif but we did uncover a typical motif for nuclear export signal (NES) as well as an EADD motif. The NES motif is embedded the codon sequence 30 PIAVDLWNV MYTLVVKYQRR 49 . The EADD motif is embedded in the codon sequence 186 INSGQLEADDACANLYHTNT 205 . The EADD motif has been reported previously as a catalytic motif is required for RNase activity (15). This motif is present in a number of cellular and phage nucleases (e.g., T5 5= exonuclease, TaqI DNA polymerase, T4 RNase H, human FEN-1) (16)(17)(18)(19)(20)(21).This report focuses on the func...

Tristetraprolin Recruits the Herpes Simplex Virion Host Shutoff RNase to AU-Rich Elements in Stress Response mRNAs To Enable Their Cleavage

“…Foremost, mRNA degradation was demonstrated in polyribosomes (28,33). In addition, the VHS mutant lacking the nuclear export signal accumulates in the nucleus.…”

“…VHS is brought into the cell during infection and is immediately transported to the nucleus either independently or bound to the U L 47 protein (28,31,32).…”

“…VHS contains a nuclear export signal and does not require the UL47 protein to shuttle from the nucleus to the cytoplasm. It requires U L 47 to shuttle from the cytoplasm to the nucleus (28).…”

Role of activating transcription factor 3 in the synthesis of latency-associated transcript and maintenance of herpes simplex virus 1 in latent state in ganglia

“…The Northern blot analyses were carried out as described previously (47). In brief, 10 μg of total RNA was loaded onto denaturing formaldehyde gel, transferred, and probed with random hexanucleotideprimed 32 P-labeled fragments of the indicated genes (ATF3 and LAT).…”

“…For this experiment, 20 μg of nuclear extracted protein was added to a binding reaction mixture containing 0.5 ng of 32 P-labeled oligonucleotide, followed by electrophoresis and autoradiography. The Oligonucleotide labeling, nuclear protein fraction extraction, and electrophoretic mobility shift assay (EMSA) were done as described previously (47)(48)(49). The canonical CRE and CRE-like (CRE1 and CRE2) oligonucleotides were designed to the LAT promoter sequence HSV-1(F) corresponding to ATF3-binding sites.…”