Shutoff Of Protein Synthesis Research Articles

Human coronavirus NL63 nsp1 induces degradation of RNA polymerase II to inhibit host protein synthesis.

Coronavirus (CoV) nonstructural protein 1 (nsp1) is considered a pathogenic factor due to its ability to inhibit host antiviral responses by inducing general shutoff of host protein synthesis. Nsp1 is expressed by α- and β-CoVs, but its functions and strategies to induce host shutoff are not fully elucidated. We compared the nsp1s from two β-CoVs (SARS-CoV and SARS-CoV-2) and two α-CoVs (NL63 and 229E) and found that NL63 nsp1 has the strongest shutoff activity. Unlike SARS-CoV nsp1s, which bind to 40S ribosomes and block translation of cellular mRNA, NL63 nsp1 did not inhibit translation of mRNAs transfected into cells. Instead, NL63 nsp1 localized to the nucleus and specifically inhibited transcription of genes under an RNA polymerase II (RNAPII) promoter. Further analysis revealed that NL63 nsp1 induces degradation of the largest subunit of RNAPII, Rpb1. This degradation was detected regardless of the phosphorylation state of Rpb1 and was blocked by the proteasome inhibitor MG132. We also found that Rpb1 was ubiquitinated in NL63-infected cells, and inhibition of ubiquitination by a ubiquitin activating enzyme inhibitor (TAK243) prevented degradation of Rpb1 in virus-infected cells. These data reveal an unrecognized strategy of host shutoff by human α-CoV NL63: targeting host transcription by inducing Rpb1 degradation to prevent host protein expression. Our study indicates that viruses within the same family can use completely distinct mechanisms to regulate host antiviral responses.

Mass-Spectrometric Evaluation of the African Swine Fever Virus-Induced Host Shutoff Using Dynamic Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC).

African swine fever is a viral disease of swine caused by the African swine fever virus (ASFV). Currently, ASFV is spreading over the Eurasian continent and threatening global pig husbandry. One viral strategy to undermine an efficient host cell response is to establish a global shutoff of host protein synthesis. This shutoff has been observed in ASFV-infected cultured cells using two-dimensional electrophoresis combined with metabolic radioactive labeling. However, it remained unclear if this shutoff was selective for certain host proteins. Here, we characterized ASFV-induced shutoff in porcine macrophages by measurement of relative protein synthesis rates using a mass spectrometric approach based on stable isotope labeling with amino acids in cell culture (SILAC). The impact of ASFV infection on the synthesis of >2000 individual host proteins showed a high degree of variability, ranging from complete shutoff to a strong induction of proteins that are absent from naïve cells. GO-term enrichment analysis revealed that the most effective shutoff was observed for proteins related to RNA metabolism, while typical representatives of the innate immune system were strongly induced after infection. This experimental setup is suitable to quantify a virion-induced host shutoff (vhs) after infection with different viruses.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

YTHDF2 Is Downregulated in Response to Host Shutoff Induced by DNA Virus Infection and Regulates Interferon-Stimulated Gene Expression.

Recent studies have begun to reveal the complex and multifunctional roles of N6-methyladenosine (m6A) modifications and their associated writer, reader, and eraser proteins in infection by diverse RNA and DNA viruses. However, little is known about their regulation and functions during infection by several viruses, including poxviruses. Here, we show that members of the YTH Domain Family (YTHDF), in particular YTHDF2, are downregulated as the prototypical poxvirus, vaccinia virus (VacV) enters later stages of replication in a variety of natural target cell types, but not in commonly used transformed cell lines wherein the control of YTHDF2 expression appears to be dysregulated. YTHDF proteins also decreased at late stages of infection by herpes simplex virus 1 (HSV-1) but not human cytomegalovirus, suggesting that YTHDF2 is downregulated in response to infections that induce host shutoff. In line with this idea, YTHDF2 was potently downregulated upon infection with a VacV mutant expressing catalytically inactive forms of the decapping enzymes, D9 and D10, which fails to degrade dsRNA and induces a protein kinase R response that itself inhibits protein synthesis. Overexpression and RNAi-mediated depletion approaches further demonstrate that YTHDF2 does not directly affect VacV replication. Instead, experimental downregulation of YTHDF2 or the related family member, YTHDF1, induces a potent increase in interferon-stimulated gene expression and establishes an antiviral state that suppresses infection by either VacV or HSV-1. Combined, our data suggest that YTHDF2 is destabilized in response to infection-induced host shutoff and serves to augment host antiviral responses. IMPORTANCE There is increasing recognition of the importance of N6-methyladenosine (m6A) modifications to both viral and host mRNAs and the complex roles this modification plays in determining the fate of infection by diverse RNA and DNA viruses. However, in many instances, the functional contributions and importance of specific m6A writer, reader, and eraser proteins remains unknown. Here, we show that natural target cells but not transformed cell lines downregulate the YTH Domain Family (YTHDF) of m6A reader proteins, in particular YTHDF2, in response to shutoff of protein synthesis upon infection with the large DNA viruses, vaccinia virus (VacV), or herpes simplex virus type 1. We further reveal that YTHDF2 downregulation also occurs as part of the host protein kinase R response to a VacV shutoff mutant and that this downregulation of YTHDF family members functions to enhance interferon-stimulated gene expression to create an antiviral state.

The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

SARS-CoV-2 Harnesses Host Translational Shutoff and Autophagy To Optimize Virus Yields: the Role of the Envelope (E) Protein.

The SARS-CoV-2 virion is composed of four structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope (E). E spans the membrane a single time and is the smallest, yet most enigmatic of the structural proteins. E is conserved among coronaviruses and has an essential role in virus-mediated pathogenesis. We found that ectopic expression of E had deleterious effects on the host cell as it activated stress responses, leading to LC3 lipidation and phosphorylation of the translation initiation factor eIF2α that resulted in host translational shutoff. During infection E is highly expressed, although only a small fraction is incorporated into virions, suggesting that E activity is regulated and harnessed by the virus to its benefit. Consistently, we found that proteins from heterologous viruses, such as the γ1 34.5 protein of herpes simplex virus 1, prevented deleterious effects of E on the host cell and allowed for E protein accumulation. This observation prompted us to investigate whether other SARS-CoV-2 structural proteins regulate E. We found that the N and M proteins enabled E protein accumulation, whereas S did not. While γ1 34.5 protein prevented deleterious effects of E on the host cells, it had a negative effect on SARS-CoV-2 replication. The negative effect of γ1 34.5 was most likely associated with failure of SARS-CoV-2 to divert the translational machinery and with deregulation of autophagy. Overall, our data suggest that SARS-CoV-2 causes stress responses and subjugates these pathways, including host protein synthesis (phosphorylated eIF2α) and autophagy, to support optimal virus replication. IMPORTANCE In late 2019, a new β-coronavirus, SARS-CoV-2, entered the human population causing a pandemic that has resulted in over 6 million deaths worldwide. Although closely related to SARS-CoV, the mechanisms of SARS-CoV-2 pathogenesis are not fully understood. We found that ectopic expression of the SARS-CoV-2 E protein had detrimental effects on the host cell, causing metabolic alterations, including shutoff of protein synthesis and mobilization of cellular resources through autophagy activation. Coexpression of E with viral proteins known to subvert host antiviral responses such as autophagy and translational inhibition, either from SARS-CoV-2 or from heterologous viruses, increased cell survival and E protein accumulation. However, such factors were found to negatively impact SARS-CoV-2 infection, as autophagy contributes to formation of viral membrane factories and translational control offers an advantage for viral gene expression. Overall, SARS-CoV-2 has evolved mechanisms to harness host functions that are essential for virus replication.

Global Transcriptome Analyses of Cellular and Viral mRNAs during HAdV-C5 Infection Highlight New Aspects of Viral mRNA Biogenesis and Cytoplasmic Viral mRNA Accumulations.

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis.

Parsing the role of NSP1 in SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1’s shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1’s explicit role in blocking the IFN response.

Rapid and quantitative evaluation of vaccinia virus-induced host shutoff using newly generated cell lines stably expressing secreted Gaussia luciferase.

Host shutoff, characterized by a global decline of cellular protein synthesis, is commonly observed in many viral infections, including vaccinia virus (VACV). Classic methods measuring host shutoff include the use of radioactive or nonradioactive probes to label newly synthesized proteins followed by radioautography or sodium dodecyl-sulfate polyacrylamide gel electrophoresisto resolve the proteins for follow-up detection. Although theseare highly reliable methods, they are time- and labor-consuming. Here, we generated two cell lines stably expressing secreted Gaussia luciferase. These reporter cells allow rapid, quantitative, and consecutive monitoring of host shutoff from a single infection sample. We evaluated host shutoff induced by wild-type and various mutant VACVs using the reporter cell lines. The results validated the utilities of the reporter cells and quantitatively characterized VACV-induced host shutoff at different stages of replication. Notably, the results also indicated additional major unidentified VACV shutoff factors. Our study provides new tools to study host shutoff. The reporter cells are also suitable for high throughput settings and rapid testing of clinically isolated viruses. In combination with classical methods, these tools will greatly facilitate understanding of virus-induced host shutoff, and protein synthesis shutoff caused by other physiologically relevant stresses.

The Mimivirus L375 Nudix enzyme hydrolyzes the 5' mRNA cap.

The giant Mimivirus is a member of the nucleocytoplasmic large DNA viruses (NCLDV), a group of diverse viruses that contain double-stranded DNA (dsDNA) genomes that replicate primarily in eukaryotic hosts. Two members of the NCLDV, Vaccinia Virus (VACV) and African Swine Fever Virus (ASFV), both synthesize Nudix enzymes that have been shown to decap mRNA, a process thought to accelerate viral and host mRNA turnover and promote the shutoff of host protein synthesis. Mimivirus encodes two Nudix enzymes in its genome, denoted as L375 and L534. Importantly, L375 exhibits sequence similarity to ASFV-DP and eukaryotic Dcp2, two Nudix enzymes shown to possess mRNA decapping activity. In this work, we demonstrate that recombinant Mimivirus L375 cleaves the 5’ m7GpppN mRNA cap, releasing m7GDP as a product. L375 did not significantly cleave mRNAs containing an unmethylated 5’GpppN cap, indicating that this enzyme specifically hydrolyzes methylated-capped transcripts. A point mutation in the L375 Nudix motif completely eliminated cap hydrolysis, showing that decapping activity is dependent on this motif. Addition of uncapped RNA significantly reduced L375 decapping activity, suggesting that L375 may recognize its substrate through interaction with the RNA body.

Influenza A viruses balance ER stress with host protein synthesis shutoff

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.

DPV UL41 gene encoding protein induces host shutoff activity and affects viral replication

The virion host shutoff (VHS) protein, encoded by the UL41 gene of herpes simplex virus (HSV), specifically degrades mRNA and induces host shutoff. VHS and its homologs are highly conserved in the Alphaherpesvirinae subfamily. However, the role of the duck plague virus (DPV) UL41 gene is unclear. In this study, we found that the DPV UL41 gene-encoded protein (pUL41) degrades RNA polymerase (pol) II-transcribed translatable RNA and induces protein synthesis shutoff. DPV pUL41 was dispensable for viral replication, but the UL41-deleted mutant virus exhibited a significant viral growth defect and plaque size reduction in Duck embryo fibroblast (DEF) cells. Furthermore, DPV pUL41 regulated viral mRNA accumulation to affect viral DNA replication, release and cell-to-cell spread.

Optimization of a GDNF production method based on Semliki Forest virus vector

Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson's disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature-dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33°C to 37°C during the “shut-off period”, produced a significant improvement in cell survival and hGDNF expression. In consonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a “recovery period” at 37°C before cells are exposed at 33°C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF.

EIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α.IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

Multiple Posttranscriptional Strategies To Regulate the Herpes Simplex Virus 1 vhs Endoribonuclease.

The herpes simplex virus 1 (HSV-1) virion host shutoff (vhs) protein is an endoribonuclease that binds to the cellular translation initiation machinery and degrades associated mRNAs, resulting in the shutoff of host protein synthesis. Hence, its unrestrained activity is considered lethal, and it has been proposed that vhs is regulated by two other virus proteins, VP22 and VP16. We have found that during infection, translation of vhs requires VP22 but not the VP22-VP16 complex. Moreover, in the absence of VP22, vhs is not overactive against cellular or viral transcripts. In transfected cells, vhs was also poorly translated, correlating with the aberrant localization of its mRNA. Counterintuitively, vhs mRNA was predominantly nuclear in cells where vhs protein was detected. Likewise, transcripts from cotransfected plasmids were also retained in the same nuclei where vhs mRNA was located, while poly(A) binding protein (PABP) was relocalized to the nucleus in a vhs-dependent manner, implying a general block to mRNA export. Coexpression of VP16 and VP22 rescued the cytoplasmic localization of vhs mRNA but failed to rescue vhs translation. We identified a 230-nucleotide sequence in the 5' region of vhs that blocked its translation and, when transferred to a heterologous green fluorescent protein transcript, reduced translation without altering mRNA levels or localization. We propose that expression of vhs is tightly regulated by a combination of inherent untranslatability and autoinduced nuclear retention of its mRNA that results in a negative feedback loop, with nuclear retention but not translation of vhs mRNA being the target of rescue by the vhs-VP16-VP22 complex.IMPORTANCE A myriad of gene expression strategies has been discovered through studies carried out on viruses. This report concerns the regulation of the HSV-1 vhs endoribonuclease, a virus factor that is important for counteracting host antiviral responses by degrading their mRNAs but that must be regulated during infection to ensure that it does not act against and inhibit the virus itself. We show that regulation of vhs involves multifaceted posttranscriptional cellular and viral processes, including aberrant mRNA localization and a novel, autoregulated negative feedback loop to target its own and coexpressed mRNAs for nuclear retention, an activity that is relieved by coexpression of two other virus proteins, VP22 and VP16. These studies reveal the interplay of strategies by which multiple virus-encoded factors coordinate gene expression at the time that they are needed. These findings are broadly relevant to both virus and cellular gene expression.

Restriction of Replication of Oncolytic Herpes Simplex Virus with a Deletion of γ34.5 in Glioblastoma Stem-Like Cells.

Oncolytic viruses, including herpes simplex viruses (HSVs), are a new class of cancer therapeutic engineered to infect and kill cancer cells while sparing normal tissue. To ensure that oncolytic HSV (oHSV) is safe in the brain, all oHSVs in clinical trial for glioma lack the γ34.5 genes responsible for neurovirulence. However, loss of γ34.5 attenuates growth in cancer cells. Glioblastoma (GBM) is a lethal brain tumor that is heterogeneous and contains a subpopulation of cancer stem cells, termed GBM stem-like cells (GSCs), that likely promote tumor progression and recurrence. GSCs and matched serum-cultured GBM cells (ScGCs), representative of bulk or differentiated tumor cells, were isolated from the same patient tumor specimens. ScGCs are permissive to replication and cell killing by oHSV with deletion of the γ34.5 genes (γ34.5- oHSV), while patient-matched GSCs were not, implying an underlying biological difference between stem and bulk cancer cells. GSCs specifically restrict the synthesis of HSV-1 true late (TL) proteins, without affecting viral DNA replication or transcription of TL genes. A global shutoff of cellular protein synthesis also occurs late after γ34.5- oHSV infection of GSCs but does not affect the synthesis of early and leaky late viral proteins. Levels of phosphorylated eIF2α and eIF4E do not correlate with cell permissivity. Expression of Us11 in GSCs rescues replication of γ34.5- oHSV. The difference in degrees of permissivity between GSCs and ScGCs to γ34.5- oHSV illustrates a selective translational regulatory pathway in GSCs that may be operative in other stem-like cells and has implications for creating oHSVs.IMPORTANCE Herpes simplex virus (HSV) can be genetically engineered to endow cancer-selective replication and oncolytic activity. γ34.5, a key neurovirulence gene, has been deleted in all oncolytic HSVs in clinical trial for glioma. Glioblastoma stem-like cells (GSCs) are a subpopulation of tumor cells thought to drive tumor heterogeneity and therapeutic resistance. GSCs are nonpermissive for γ34.5- HSV, while non-stem-like cancer cells from the same patient tumors are permissive. GSCs restrict true late protein synthesis, despite normal viral DNA replication and transcription of all kinetic classes. This is specific for true late translation as early and leaky late transcripts are translated late in infection, notwithstanding shutoff of cellular protein synthesis. Expression of Us11 in GSCs rescues the replication of γ34.5- HSV. We have identified a cell type-specific innate response to HSV-1 that limits oncolytic activity in glioblastoma.

Abstract P1-04-01: A novel oncolytic herpes simplex virus, GD116, has enhanced antitumour efficacy in human breast cancer

Abstract Background: Oncolytic herpes simplex virus-1 (oHSV-1) vectors are promising new therapeutic agents for cancer. For the nuerovirulence of ICP34.5 protein, the encoding gene named γ34.5 is deleted in all oHSV-1 mutants currently in clinical trials. However, the deletion of γ34.5 also promotes the interferon-induced shutoff of virual protein synthesis, and attenuates the virus replication in cancer cells at the same time. Previous studies reported that the carboxyl-terminus of MyD116/GADD34 was homologous to that of γ34.5 gene; hence, it might substitute for γ34.5 gene to enhance the replication and cytotoxicity of the virus. Objective: To reconstruct a novel oHSV-1 mutant named GD116 by inserting γ34.5-MyD116 chimeric gene into G47Δ genome, and to investigate whether the carboxyl-terminus of murine MyD116 can enhance the antitumour efficacy of GD116 on human breast cancer cell line in vitro and in vivo. Methods: Using G47Δ genome and bacterial artificial chromesome (BAC) as the backbone, the GD116 virus expressing γ34.5-MyD116 was reconstructed via Cre/loxp and FLP/FRT recombinase systems. A GD-empty virus mutant containing only the CMV sequence was also constructed as a control at the same time. Then, the replication and cytotoxicity of these two virus mutants in MDA-MB-231 humanbreast cancer cell line was evaluated in vitro. Meanwhile, the subcutaneous tumor model was also generated by injecting MDA-MB-231 cells into the right flank of BALB/c mice. Then, the antitumor efficacy of GD116 on breast cancer in vivo was evaluated by intratumoral injection. Results: Compared with GD-empty, GD116 possessed an enhanced replication capability and oncolytic efficacy in MDA-MB-231 cell line. On the fifth day after infection with GD116 at MOIs of 0.1 and 0.3, 35.0% and 50.2% of MDA-MB-231 cells were killed respectively, which were higher than that with GD-empty (24.3% and 37.8%). Additionally, GD116 exhibited enhanced antitumor efficacy on subcutaneous tumor xenografts of human breast cancer cells in vivo compared with GD-empty. On day 21 after treatment, the average tumor volume of mice was 585.5 mm3, 289.0 mm3 and 121.9 mm3 in Control, GD-empty and GD116 group respectively. Conclusion: Our findings indicate that the carboxyl-terminus of the murine MyD116 can substitute for the corresponding domain of the γ34.5 gene in oHSV-1 to promote the replication of the virus in infected cancer cells; and the mutant GD116 armed with γ34.5-MyD116 chimaera has enhanced oncolytic effect on MDA-MB-231 breast cancer cells in vitro and in vivo. Citation Format: Cheng L, Wang J, Liu R. A novel oncolytic herpes simplex virus, GD116, has enhanced antitumour efficacy in human breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-04-01.

Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I-mediated immunity.

The sensor retinoic acid-inducible gene-I (RIG-I) detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to play a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during DNA virus infection are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bind to RIG-I during herpes simplex virus 1 (HSV-1) infection. HSV-1 infection induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated RNA5SP141-interacting proteins, thereby allowing RNA5SP141 to bind RIG-I and induce type I interferon. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related Epstein-Barr virus (EBV) as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following viral depletion of their respective binding proteins.

Foot-and-mouth disease virus induces lysosomal degradation of host protein kinase PKR by 3C proteinase to facilitate virus replication

The interferon-induced double-strand RNA activated protein kinase (PKR) plays important roles in host defense against viral infection. Here we demonstrate the significant antiviral role of PKR against foot-and-mouth disease virus (FMDV) and report that FMDV infection inhibits PKR expression and activation in porcine kidney (PK-15) cells. The viral nonstructural protein 3C proteinase (3Cpro) is identified to be responsible for this inhibition. However, it is independent of the well-known proteinase activity of 3Cpro or 3Cpro-induced shutoff of host protein synthesis. We show that 3Cpro induces PKR degradation by lysosomal pathway and no interaction is determined between 3Cpro and PKR. Together, our results indicate that PKR acts an important antiviral factor during FMDV infection, and FMDV has evolved a strategy to overcome PKR-mediated antiviral role by downregulation of PKR protein.

PA-X protein contributes to virulence of triple-reassortant H1N2 influenza virus by suppressing early immune responses in swine

Previous studies have identified a functional role of PA-X for influenza viruses in mice and avian species; however, its role in swine remains unknown. Toward this, we constructed PA-X deficient virus (Sw-FS) in the background of a Triple-reassortment (TR) H1N2 swine influenza virus (SIV) to assess the impact of PA-X in viral virulence in pigs. Expression of PA-X in TR H1N2 SIV enhanced viral replication and host protein synthesis shutoff, and inhibited the mRNA levels of type I IFNs and proinflammatory cytokines in porcine cells. A delay of proinflammatory responses was observed in lungs of pigs infected by wild type SIV (Sw-WT) compared to Sw-FS. Furthermore, Sw-WT virus replicated and transmitted more efficiently than Sw-FS in pigs. These results highlight the importance of PA-X in the moderation of virulence and immune responses of TR SIV in swine, which indicated that PA-X is a pro-virulence factor in TR SIV in pigs.