Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions.

There is an outstanding need for broadly acting antiviral drugs to combat emerging viral diseases. Here, we report that thiopurines inhibit the replication of the betacoronaviruses HCoV-OC43 and SARS-CoV-2. 6-Thioguanine (6-TG) disrupted early stages of infection, limiting accumulation of full-length viral genomes, subgenomic RNAs and structural proteins. In ectopic expression models, we observed that 6-TG increased the electrophoretic mobility of Spike from diverse betacoronaviruses, matching the effects of enzymatic removal of N-linked oligosaccharides from Spike in vitro. SARS-CoV-2 virus-like particles (VLPs) harvested from 6-TG-treated cells were deficient in Spike. 6-TG treatment had a similar effect on production of lentiviruses pseudotyped with SARS-CoV-2 Spike, yielding pseudoviruses deficient in Spike and unable to infect ACE2-expressing cells. Together, these findings from complementary ectopic expression and infection models strongly indicate that defective Spike trafficking and processing is an outcome of 6-TG treatment. Using biochemical and genetic approaches we demonstrated that 6-TG is a pro-drug that must be converted to the nucleotide form by hypoxanthine phosphoribosyltransferase 1 (HPRT1) to achieve antiviral activity. This nucleotide form has been shown to inhibit small GTPases Rac1, RhoA, and CDC42; however, we observed that selective chemical inhibitors of these GTPases had no effect on Spike processing or accumulation. By contrast, the broad GTPase agonist ML099 countered the effects of 6-TG, suggesting that the antiviral activity of 6-TG requires the targeting of an unknown GTPase. Overall, these findings suggest that small GTPases are promising targets for host-targeted antivirals.

Thiopurines activate an antiviral unfolded protein response that blocks influenza A virus glycoprotein accumulation.

Influenza A viruses (IAVs) utilize host shutoff mechanisms to limit antiviral gene expression and redirect translation machinery to the synthesis of viral proteins. Previously, we showed that IAV replication is sensitive to protein synthesis inhibitors that block translation initiation and induce formation of cytoplasmic condensates of untranslated messenger ribonucleoprotein complexes called stress granules (SGs). In this study, using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that triggered SG formation in IAV-infected cells and blocked IAV replication in a dose-dependent manner without eliciting SG formation in uninfected cells. 6-TG and 6-TGo selectively disrupted the synthesis and maturation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA) without affecting the levels of the viral RNAs that encode them. By contrast, these thiopurines had minimal effect on other IAV proteins or the global host protein synthesis. Disruption of IAV glycoprotein accumulation by 6-TG and 6-TGo correlated with activation of unfolded protein response (UPR) sensors activating transcription factor-6 (ATF6), inositol requiring enzyme-1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), leading to downstream UPR gene expression. Treatment of infected cells with the chemical chaperone 4-phenylbutyric acid diminished thiopurine-induced UPR activation and partially restored the processing and accumulation of HA and NA. By contrast, chemical inhibition of the integrated stress response downstream of PERK restored accumulation of NA monomers but did not restore processing of viral glycoproteins. Genetic deletion of PERK enhanced the antiviral effect of 6-TG without causing overt cytotoxicity, suggesting that while UPR activation correlates with diminished viral glycoprotein accumulation, PERK could limit the antiviral effects of drug-induced ER stress. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and selectively disrupt accumulation of viral glycoproteins.IMPORTANCESecreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. Many viruses rely on the ER for the synthesis and processing of viral glycoproteins that will ultimately be incorporated into viral envelopes. Viral burden on the ER can trigger the unfolded protein response (UPR). Much remains to be learned about how viruses co-opt the UPR to ensure efficient synthesis of viral glycoproteins. Here, we show that two FDA-approved thiopurine drugs, 6-TG and 6-TGo, induce the UPR, which represents a previously unrecognized effect of these drugs on cell physiology. This thiopurine-mediated UPR activation blocks influenza virus replication by impeding viral glycoprotein accumulation. Our findings suggest that 6-TG and 6-TGo may have broad antiviral effect against enveloped viruses that require precise tuning of the UPR to support viral glycoprotein synthesis.

Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins.

Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-ß and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity.IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

Adaptive Mutations in Influenza A/California/07/2009 Enhance Polymerase Activity and Infectious Virion Production.

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (also known as CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering three PB1 and PA mutations into the parental CA/07 strain, we demonstrated that these mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

Taxonomic differences of gut microbiomes drive cellulolytic enzymatic potential within hind-gut fermenting mammals.

Host diet influences the diversity and metabolic activities of the gut microbiome. Previous studies have shown that the gut microbiome provides a wide array of enzymes that enable processing of diverse dietary components. Because the primary diet of the porcupine, Erethizon dorsatum, is lignified plant material, we reasoned that the porcupine microbiome would be replete with enzymes required to degrade lignocellulose. Here, we report on the bacterial composition in the porcupine microbiome using 16S rRNA sequencing and bioinformatics analysis. We extended this analysis to the microbiomes of 20 additional mammals located in Shubenacadie Wildlife Park (Nova Scotia, Canada), enabling the comparison of bacterial diversity amongst three mammalian taxonomic orders (Rodentia, Carnivora, and Artiodactyla). 16S rRNA sequencing was validated using metagenomic shotgun sequencing on selected herbivores (porcupine, beaver) and carnivores (coyote, Arctic wolf). In the microbiome, functionality is more conserved than bacterial composition, thus we mined microbiome data sets to identify conserved microbial functions across species in each order. We measured the relative gene abundances for cellobiose phosphorylase, endoglucanase, and beta-glucosidase to evaluate the cellulose-degrading potential of select mammals. The porcupine and beaver had higher proportions of genes encoding cellulose-degrading enzymes than the Artic wolf and coyote. These findings provide further evidence that gut microbiome diversity and metabolic capacity are influenced by host diet.

Stress Granule-Inducing Eukaryotic Translation Initiation Factor 4A Inhibitors Block Influenza A Virus Replication.

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5' untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication.

Molecular characterization of pathogenic and nonpathogenic fowl aviadenovirus serotype 11 isolates.

Fowl aviadenoviruses, many of which are of importance in veterinary medicine, are classified into 5 species. In this study, a pathogenic isolate and a nonpathogenic isolate of fowl aviadenovirus serotype 11 (FAdV-11) of species Fowl aviadenovirus D were characterized. Growth rates were analyzed for the 2 isolates, showing notable differences. The complete genomic sequences of the viruses were fully determined and were analyzed. The genomes of the 2 isolates showed 98.1% sequence identity and revealed 6 nonsynonymous mutations between the Ontario isolates. Two of the 6 mutations were also found in the sequences of recently published pathogenic Chinese fowl aviadenovirus 11 isolates, suggesting potential molecular markers that could be associated with pathogenesis. Deletions were found in the L5 region within the overlapping coding sequences for the 100, 22, and 33 kDa proteins, and these were found in only the nonpathogenic isolates. This molecular pattern was identified in FAdV-9, another nonpathogenic FAdV-D species virus. Furthermore, the tandem repeat regions varied dramatically; the pathogenic isolates contained a reduced number of tandem repeats compared with the nonpathogenic isolates. Lastly, a protein produced early in infection was analyzed using bioinformatics to determine its role in disease. This study highlights several candidate molecular determinants of avian adenovirus genomes related to pathogenicity.