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Hepatitis B virus (HBV) mRNA metabolism is dependent upon host proteins PAPD5 and PAPD7 (PAPD5/7). PAPD5/7 are cellular, noncanonical, poly(A) polymerases (PAPs) whose main function is to oligoadenylate the 3′ end of noncoding RNA (ncRNA) for exosome degradation. HBV seems to exploit these two ncRNA quality-control factors for viral mRNA stabilization, rather than degradation. RG7834 is a small-molecule compound that binds PAPD5/7 and inhibits HBV gene production in both tissue culture and animal study. We reported that RG7834 was able to destabilize multiple HBV mRNA species, ranging from the 3.5-kb pregenomic/precore mRNAs to the 2.4/2.1-kb hepatitis B virus surface protein (HBs) mRNAs, except for the smallest 0.7-kb X protein (HBx) mRNA. Compound-induced HBV mRNA destabilization was initiated by a shortening of the poly(A) tail, followed by an accelerated degradation process in both the nucleus and cytoplasm. In cells expressing HBV mRNA, both PAPD5/7 were found to be physically associated with the viral RNA, and the polyadenylating activities of PAPD5/7 were susceptible to RG7834 repression in a biochemical assay. Moreover, in PAPD5/7 double-knockout cells, viral transcripts with a regular length of the poly(A) sequence could be initially synthesized but became shortened in hours, suggesting that participation of PAPD5/7 in RNA 3′ end processing, either during adenosine oligomerization or afterward, is crucial for RNA stabilization.

RNA-binding proteins (RBPs) play a major role during control of mRNA localization, stability, and translation and are central to most cellular processes. In the fission yeast Schizosaccharomyces pombe, the multiple K homology (KH) domain RBP Rnc1 downregulates the activity of the cell integrity pathway (CIP) via stabilization of pmp1+ mRNA, which encodes the Pmp1 phosphatase that inactivates Pmk1, the mitogen-activated protein kinase (MAPK) component of this signaling cascade. However, Rnc1 likely regulates the half-life/stability of additional mRNAs. We show that Rnc1 downregulates the activity of Sty1, the MAPK of the stress-activated MAPK pathway (SAPK), during control of cell length at division and recovery in response to acute stress. Importantly, this control strictly depends on Rnc1’s ability to bind mRNAs encoding activators (Wak1 MAPKKK, Wis1 MAPKK) and downregulators (Atf1 transcription factor, Pyp1 and Pyp2 phosphatases) of Sty1 phosphorylation through its KH domains. Moreover, Sty1 is responsible for Rnc1 phosphorylation in vivo at multiple phosphosites during growth and stress, and these modifications trigger Rnc1 for proper binding and destabilization of the above mRNA targets. Phosphorylation by Sty1 prompts Rnc1-dependent mRNA destabilization to negatively control SAPK signaling, thus revealing an additional feedback mechanism that allows precise tuning of MAPK activity during unperturbed cell growth and stress., This work was supported by the Ministerio de Ciencia, Innovación y Universidades, Spain (grant reference BFU2017-82423-P to J.C.) and European Regional Development Fund (ERDF) cofunding from the European Union.

The herpes simplex virus 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase that promotes the degradation of several host cell proteins. Most studies have found that ICP0 promotes the loss of IFI16 in infected cells, but one study reported that ICP0 was not necessary or sufficient for loss of IFI16 in a tumor-derived cell line. Therefore, in this study, we examined the requirement for ICP0 in promoting the loss of IFI16 in several normal and tumor-derived cell lines. HSV-1 infection resulted in an observable decrease of IFI16 protein levels in normal human foreskin fibroblasts (HFFs), normal oral keratinocytes (NOKs), and HeLa cells but not in U2OS cells. During infection with an ICP0-null virus, we observed a reduced loss of IFI16 in HFFs and NOKs but not in HeLa cells. Ectopic expression of ICP0 from a transfected plasmid was sufficient to promote the loss of IFI16 in HFFs and NOKs. In the absence of ICP0, we observed a delayed reduction of IFI16 protein that correlated with a reduction in the steady-state levels of IFI16 mRNA. In addition, we show that the ICP0-independent loss of IFI16 in HeLa cells is dependent in part on the activity of the viral virion host shutoff (vhs) tegument protein. Together, these results demonstrate that HSV-1 promotes the loss of IFI16 through at least two mechanisms: (i) by ICP0-dependent degradation of IFI16 and (ii) by vhs-dependent turnover of IFI16 mRNA. In addition, this study highlights a potential intrinsic difference between normal and tumor-derived cells for the activities of IFI16 and HSV-1 ICP0. IMPORTANCE HSV-1 is a ubiquitous virus that establishes a lifetime persistent infection in humans. The relative success of HSV-1 as a pathogen is, in part, dependent on the expression of viral proteins that counteract host intrinsic defense mechanisms and that modulate immune responses during viral infection. In this study, we examined the relative roles of two viral gene products for the ability to promote loss of the antiviral IFI16 DNA sensor. We demonstrate that the viral immediate early ICP0 protein plays a dominant role in the loss of IFI16 in normal, but not tumor-derived, human cell lines. In contrast, viral vhs-mediated loss of IFI16 by mRNA destabilization is revealed to be dominant in tumor-derived cells in which ICP0 is nonfunctional. Together, these results contribute to our understanding of how HSV-1 modulates IFI16 protein levels and highlight cell-type-dependent differences between normal and tumor-derived cells.

We have previously shown that herpes simplex virus type 1 (HSV-1) infection is associated with early destabilization/degradation of infected cell mRNAs and consequent shutoff of host protein synthesis by the activity of the virion-associated host shutoff (vhs) UL41 protein. Wild-type (wt) virus destabilized/degraded the housekeeping β-actin and α-tubulin mRNAs as well host stress functions, like the heat shock 70 protein induced postinfection. vhs mutants did not degrade the mRNAs. Elaborate studies by others have been concerned with the mode of mRNA degradation and the mRNAs affected. We now describe vhs activity in primary cultures of mouse cerebellar granule neurons (CGNs). Specifically, (i) upon infection in the presence of actinomycin D to test activity of input viral particles, there was a generalized inhibition of protein synthesis, which depended on the input multiplicity of infection (MOI). (ii) Low-MOI infection with vhs-1 mutant virus was associated with increased synthesis of all apparent proteins. Higher MOIs caused some shutoff, albeit significantly lower than that of wt virus. This pattern could reflect an interaction(s) of vhs-1 protein with host machinery involved in cellular mRNA destabilization/degradation, sequestering this activity. (iii) wt virus infection was associated with cell survival, at least for a while, whereas mutant virus induced apoptotic cell death at earlier times. (iv) wt virus replicated well in the CGNs, whereas there was no apparent replication of the vhs-1 mutant virus. (v) The vhs-1 mutant could serve as helper virus for composite amplicon vectors carrying marker genes and the human p53 gene. Ongoing studies test the use of vhs-1-based composite oncolytic vectors towards cancer gene therapy.

Expression of the Anabaena sp. strain PCC 7120 RNA helicase gene crhC is induced by cold shock. crhC transcripts are not detectable at 30°C but accumulate at 20°C, and levels remain elevated for the duration of the cold stress. Light-derived metabolic capability, and not light per se, is required for crhC transcript accumulation. Enhanced crhC mRNA stability contributes significantly to the accumulation of crhC transcripts, with the crhC half-life increasing sixfold at 20°C. The accumulation is reversible, with the cells responding more rapidly to temperature downshifts than to upshifts, as a result of the lack of active mRNA destabilization and the continuation of crhC transcription, at least transiently, after a temperature upshift. Translational inhibitors do not induce crhC expression to cold shock levels, indicating that inhibition of translation is only one of the signals required to activate the cold shock response in Anabaena . Limited amounts of protein synthesis are required for the cold shock-induced accumulation of crhC transcripts, as normal levels of accumulation occur in the presence of tetracycline but are abolished by chloramphenicol. Regulation of crhC expression may also extend to the translational level, as CrhC protein levels do not correlate completely with the pattern of mRNA transcript accumulation. Our experiments indicate that the regulation of crhC transcript accumulation is tightly controlled by both temperature and metabolic activity at the levels of transcription, mRNA stabilization, and translation.

Three transcripts from the terminal repeat of the channel catfish virus (CCV; also known as ictalurid herpesvirus 1) genome were mapped by S1 nuclease and primer extension analyses as well as by cDNA sequencing. These transcripts, TR3 , TR5/6 , and TR6 , are encoded by open reading frame (ORF) 3, ORFs 5 and 6, and ORF 6, respectively, and correspond to those previously identified by sequence analysis (A. J. Davison, Virology 186:9–14, 1992). ORF 5 has previously been determined to encode thymidine kinase, but ORF 3 and ORF 6 encode proteins of unknown function. Although all three transcripts accumulate to high levels in cells infected in the presence of cycloheximide, kinetic analysis demonstrates that TR5/6 and TR6 are either early or late transcripts that leak through the cycloheximide block. In addition, two transcripts from the terminal repeat of the CCV genome that were mapped previously and were thought to be immediate-early in character, TR8a/9 and TR9 , exhibit kinetics characteristic of early or late transcripts. TR3 is an immediate-early transcript that appears to have a very short half-life. In the 3′ untranslated region of TR3 , there are three copies of an AU-rich element which has previously been shown to be involved in destabilization of the oncogene c- fos and granulocyte/macrophage colony-stimulating factor mRNAs. mRNA destabilization may represent another mechanism by which herpesviruses regulate the rapid switch in expression from immediate-early genes to early genes during the transition to the early phase of infection.

Posttranscriptional control of nif mRNA stability was demonstrated by functional and chemical analyses, using specific probes for four nif transcripts. In the wild type, nif transcripts (except nifLA) were stable during derepression, with half-lives of approximately 30 min. They were dramatically destabilized by O2 or elevated temperature (41 degrees C) and to a lesser extent by NH4+. In contrast, the nifLA message was not particularly stable, and posttranscriptional control was not evident. In NifL- strains, both forms of analysis indicated that the nifL product was involved in nif mRNA destabilization in the presence of O2 and NH4+.