The RNA Interference Effector Protein Argonaute 2 Functions as a Restriction Factor Against SARS-CoV-2.

Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then degraded or translationally suppressed. In plants and invertebrates, the RNAi pathway has well-described roles in antiviral defense, but its function in limiting viral infections in mammalian cells is less well understood. Here, we examined the role of Ago2 in replication of the betacoronavirus SARS-CoV-2, the etiologic agent of COVID-19. Microscopic analyses of infected cells revealed that a pool of Ago2 closely associates with viral replication sites and gene ablation studies showed that loss of Ago2 resulted in over 1,000-fold increase in peak viral titers. Replication of the alphacoronavirus 229E was also significantly increased in cells lacking Ago2. The antiviral activity of Ago2 was dependent on both its ability to bind small RNAs and its endonuclease function. Interestingly, in cells lacking Dicer, an upstream component of the RNAi pathway, viral replication was the same as in parental cells. This suggests that the antiviral activity of Ago2 is independent of Dicer processed miRNAs. Deep sequencing of infected cells by other groups identified several SARS-CoV-2-derived small RNAs that bind to Ago2. A mutant virus lacking the most abundant ORF7A-derived viral miRNA was found to be significantly less sensitive to Ago2-mediated restriction. This combined with our findings that endonuclease and small RNA-binding functions of Ago2 are required for its antiviral function, suggests that Ago2-small viral RNA complexes target nascent viral RNA produced at replication sites for cleavage. Further studies are required to elucidate the processing mechanism of the viral small RNAs that are used by Ago2 to limit coronavirus replication.

Identification of Human Host Substrates of the SARS-CoV-2 Mpro and PLpro Using Subtiligase N-Terminomics.

The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, Mpro and PLpro, that are thought to play key roles in the suppression of host protein synthesis and immune response evasion during infection. To identify the specific host cell substrates of these proteases, active recombinant SARS-CoV-2 Mpro and PLpro were added to A549 and Jurkat human cell lysates, and subtiligase-mediated N-terminomics was used to capture and enrich protease substrate fragments. The precise location of each cleavage site was identified using mass spectrometry. Here, we report the identification of over 200 human host proteins that are potential substrates for SARS-CoV-2 Mpro and PLpro and provide a global mapping of proteolysis for these two viral proteases in vitro. Modulating proteolysis of these substrates will increase our understanding of SARS-CoV-2 pathobiology and COVID-19.

Flavivirus Capsid Proteins Inhibit the Interferon Response.

Zika virus (ZIKV) establishes persistent infections in multiple human tissues, a phenomenon that likely plays a role in its ability to cause congenital birth defects and neurological disease. Multiple nonstructural proteins encoded by ZIKV, in particular NS5, are known to suppress the interferon (IFN) response by attacking different steps in this critical antiviral pathway. Less well known are the potential roles of structural proteins in affecting the host immune response during ZIKV infection. Capsid proteins of flaviviruses are of particular interest because a pool of these viral proteins is targeted to the nuclei during infection and, as such, they have the potential to affect host cell gene expression. In this study, RNA-seq analyses revealed that capsid proteins from six different flaviviruses suppress expression of type I IFN and IFN-stimulated genes. Subsequent interactome and in vitro ubiquitination assays showed that ZIKV capsid protein binds to and prevents activating ubiquitination of RIG-I CARD domains by TRIM25, a host factor that is important for the induction arm of the IFN response. The other flavivirus capsid proteins also interacted with TRIM25, suggesting that these viral proteins may attenuate antiviral signaling pathways at very early stages of infection, potentially even before nonstructural proteins are produced.

Mayaro Virus Non-Structural Protein 2 Circumvents the Induction of Interferon in Part by Depleting Host Transcription Initiation Factor IIE Subunit 2.

Mayaro virus (MAYV) is an emerging mosquito-transmitted virus that belongs to the genus Alphavirus within the family Togaviridae. Humans infected with MAYV often develop chronic and debilitating arthralgia and myalgia. The virus is primarily maintained via a sylvatic cycle, but it has the potential to adapt to urban settings, which could lead to large outbreaks. The interferon (IFN) system is a critical antiviral response that limits replication and pathogenesis of many different RNA viruses, including alphaviruses. Here, we investigated how MAYV infection affects the induction phase of the IFN response. Production of type I and III IFNs was efficiently suppressed during MAYV infection, and mapping revealed that expression of the viral non-structural protein 2 (nsP2) was sufficient for this process. Interactome analysis showed that nsP2 interacts with DNA-directed RNA polymerase II subunit A (Rpb1) and transcription initiation factor IIE subunit 2 (TFIIE2), which are host proteins required for RNA polymerase II-mediated transcription. Levels of these host proteins were reduced by nsP2 expression and during infection by MAYV and related alphaviruses, suggesting that nsP2-mediated inhibition of host cell transcription is an important aspect of how some alphaviruses block IFN induction. The findings from this study may prove useful in design of vaccines and antivirals, which are currently not available for protection against MAYV and infection by other alphaviruses.

CRISPR Technique Incorporated with Single-Cell RNA Sequencing for Studying Hepatitis B Infection.

Single-cell RNA sequencing (scRNA-seq) provides rich transcriptomic information for studying molecular events and cell heterogeneity at the single-cell level. However, it is challenging to obtain sequence information from rare or low-abundance genes in the presence of other highly abundant genes. We report here a CRISPR-Cas9 technique for the depletion of high-abundance transcripts, resulting in preferential enrichment of rare transcripts. We demonstrate an application of this CRISPR-mediated enrichment technique to scRNA-seq of liver cells infected with hepatitis B virus (HBV). Direct sequencing without the CRISPR-mediated enrichment detected HBV RNA in only 0.6% of the cells. The CRISPR-mediated depletion of the three most abundant transcripts resulted in selective enrichment of the HBV transcript and successful sequencing of HBV RNA in more than 74% of the cells. The improvement enabled a study of HBV infection and interferon treatment of a liver cell model. Gene clusters between the control and HBV-infected Huh7.5-NTCP cells were similar, suggesting that HBV infection did not significantly alter gene expression of the host cells. The treatment with interferon alpha dramatically changed the gene expression of Huh7.5-NTCP cells. These results from the single cell RNA-seq analysis of 7370 cells are consistent with those of bulk experiments, suggesting that HBV is a "stealth virus".

SARS-CoV-2 Nonstructural Protein 1 Inhibits the Interferon Response by Causing Depletion of Key Host Signaling Factors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. While previous studies have shown that several SARS-CoV-2 proteins can antagonize the interferon (IFN) response, some of the mechanisms by which they do so are not well understood. In this study, we describe two novel mechanisms by which SARS-CoV-2 blocks the IFN pathway. Type I IFNs and IFN-stimulated genes (ISGs) were poorly induced during SARS-CoV-2 infection, and once infection was established, cells were highly resistant to ectopic induction of IFNs and ISGs. Levels of two key IFN signaling pathway components, Tyk2 and STAT2, were significantly lower in SARS-CoV-2-infected cells. Expression of nonstructural protein 1 (NSP1) or nucleocapsid in the absence of other viral proteins was sufficient to block IFN induction, but only NSP1 was able to inhibit IFN signaling. Mapping studies suggest that NSP1 prevents IFN induction in part by blocking IRF3 phosphorylation. In addition, NSP1-induced depletion of Tyk2 and STAT2 dampened ISG induction. Together, our data provide new insights into how SARS-CoV-2 successfully evades the IFN system to establish infection. IMPORTANCE SARS-CoV-2 is the causative agent of COVID-19, a serious disease that can have a myriad of symptoms from loss of taste and smell to pneumonia and hypercoagulation. The rapid spread of SARS-CoV-2 can be attributed in part to asymptomatic transmission, where infected individuals shed large amounts of virus before the onset of disease. This is likely due to the ability of SARS-CoV-2 to effectively suppress the innate immune system, including the IFN response. Indeed, we show that the IFN response is efficiently blocked during SARS-CoV-2 infection, a process that is mediated in large part by nonstructural protein 1 and nucleocapsid. Our study provides new insights on how SARS-CoV-2 evades the IFN response to successfully establish infection. These findings should be considered for the development and administration of therapeutics against SARS-CoV-2.

Glycomic analysis of host response reveals high mannose as a key mediator of influenza severity.

Influenza virus infections cause a wide variety of outcomes, from mild disease to 3 to 5 million cases of severe illness and ∼290,000 to 645,000 deaths annually worldwide. The molecular mechanisms underlying these disparate outcomes are currently unknown. Glycosylation within the human host plays a critical role in influenza virus biology. However, the impact these modifications have on the severity of influenza disease has not been examined. Herein, we profile the glycomic host responses to influenza virus infection as a function of disease severity using a ferret model and our lectin microarray technology. We identify the glycan epitope high mannose as a marker of influenza virus-induced pathogenesis and severity of disease outcome. Induction of high mannose is dependent upon the unfolded protein response (UPR) pathway, a pathway previously shown to associate with lung damage and severity of influenza virus infection. Also, the mannan-binding lectin (MBL2), an innate immune lectin that negatively impacts influenza outcomes, recognizes influenza virus-infected cells in a high mannose-dependent manner. Together, our data argue that the high mannose motif is an infection-associated molecular pattern on host cells that may guide immune responses leading to the concomitant damage associated with severity.

Fibroblast Growth Factor 2 Enhances Zika Virus Infection in Human Fetal Brain.

Zika virus (ZIKV) is an emerging pathogen that can cause microcephaly and other neurological defects in developing fetuses. The cellular response to ZIKV in the fetal brain is not well understood. Here, we show that ZIKV infection of human fetal astrocytes (HFAs), the most abundant cell type in the brain, results in elevated expression and secretion of fibroblast growth factor 2 (FGF2). This cytokine was shown to enhance replication and spread of ZIKV in HFAs and human fetal brain explants. The proviral effect of FGF2 is likely mediated in part by suppression of the interferon response, which would represent a novel mechanism by which viruses antagonize host antiviral defenses. We posit that FGF2-enhanced virus replication in the fetal brain contributes to the neurodevelopmental disorders associated with in utero ZIKV infection. As such, targeting FGF2-dependent signaling should be explored further as a strategy to limit replication of ZIKV.

Human Sertoli cells support high levels of Zika virus replication and persistence.

Zika virus is a teratogenic mosquito-transmitted flavivirus that is associated with birth defects in newborns and Guillain-Barré syndrome in adults. The virus can also be sexually transmitted, but currently, very little is known about the cell types supporting virus replication and persistence in human testes. Using primary cell cultures, we observed that Sertoli but not Leydig cells are highly susceptible to Zika virus infection, a process that is dependent on the TAM family receptor Axl. In cell culture, Sertoli cells could be productively infected with Zika virus for at least 6-weeks. Infection of Sertoli cells resulted in dramatic changes to the transcriptional profile of these cells. The most upregulated mRNA in infected cells was basic fibroblast growth factor (FGF2), a cytokine that was found to enhance Zika virus replication and support viral persistence. Together these findings provide key insights into understanding how Zika virus persists in the male reproductive tract and in turn may aid in developing antiviral therapies or strategies to minimize sexual transmission of this pathogen.

Expression of flavivirus capsids enhance the cellular environment for viral replication by activating Akt-signalling pathways.

Flaviviruses depend on multiple host pathways during their life cycles and have evolved strategies to avoid the innate immune response. Previously, we showed that the West Nile virus capsid protein plays a role in this process by blocking apoptosis. In this study, we examined how expression of capsid proteins from several flaviviruses affects apoptosis and other host processes that impact virus replication. All of the tested capsid proteins protected cells from Fas-dependent apoptosis through a mechanism that requires activated Akt. Capsid expression upregulated other Akt-dependent cellular processes including expression of glucose transporter 1 and mitochondrial metabolism. Protein phosphatase 1, which is known to inactivate Akt, was identified as a DENV capsid interacting protein. This suggests that DENV capsid expression activates Akt by sequestering phosphatases that downregulate phospho-Akt. Capsid-dependent upregulation of Akt would enhance downstream signalling pathways that affect cell survival and metabolism, thus providing a favourable environment for virus replication.

Functional analyses of phosphorylation events in human Argonaute 2.

Argonaute 2 (Ago2) protein is a central effector of RNA interference (RNAi) pathways and regulates mammalian genes on a global level. The mechanisms of Ago2-mediated silencing are well understood, but less is known about its regulation. Recent reports indicate that phosphorylation significantly affects Ago2 activity. Here, we investigated the effect of mutating all known phospho-residues within Ago2 on its localization and activity. Ago2 associates with two different cytoplasmic RNA granules known as processing bodies (P-bodies) and stress granules, but the nature of this phenomenon is controversial. We report that replacing serine with a phospho-mimetic aspartic acid at position 798 completely abrogates association of Ago2 with P-bodies and stress granules. The effect of this mutation on its activity in gene silencing was modest, which was surprising because association of Ago2 with cytoplasmic RNA granules is thought to be a consequence of its role in RNAi. As such, our data indicate that targeting of Ago2 to P-bodies and stress granules is separable from its role in RNAi and likely requires dynamic phosphorylation of serine 798.

MicroRNA-binding is required for recruitment of human Argonaute 2 to stress granules and P-bodies.

Argonaute proteins are the core components of the RNA-induced silencing complex, the central effector of the mammalian RNA interference pathway. In the cytoplasm, they associate with at least two types of cytoplasmic RNA granules; processing bodies and stress granules, which function in mRNA degradation and translational repression, respectively. The significance of Argonaute association with these RNA granules is not entirely clear but it is likely related to their activities within the RNAi pathway. Understanding what regulates targeting of Argonautes to RNA granules may provide clues as to their functions at these organelles. To this end, there are a number of conflicting reports that describe the role of small RNAs in targeting Argonaute proteins in mammalian cells. We employed quantitative microscopic analyses of human Argonaute 2 (hAgo2) mutants to study factors that govern localization of this RNA-binding protein to cytoplasmic RNA granules. We report, for the first time, that hAgo2 is recruited to stress granules as a consequence of its interaction with miRNAs. Moreover, loading of small RNAs onto hAgo2 is not required for its stability, suggesting that a pool of unloaded hAgo2 may exist for extended periods of time in the cytoplasm.

Live cell imaging of Argonaute proteins in mammalian cells.

The central effector of mammalian RNA interference (RNAi) is the RNA-induced silencing complex (RISC). Proteins of the Argonaute family are the core components of RISC. Recent work from multiple laboratories has shown that Argonaute family members are associated with at least two types of cytoplasmic RNA granules: GW/Processing bodies and stress granules. These Argonaute-containing granules harbor proteins that function in mRNA degradation and translational repression in response to stress. The known role of Argonaute proteins in miRNA-mediated translational repression and siRNA-directed mRNA cleavage (i.e., Argonaute 2) has prompted speculation that the association of Argonautes with these granules may reflect the activity of RNAi in vivo. Accordingly, studying the dynamic association between Argonautes and RNA granules in living cells will undoubtedly provide insight into the regulatory mechanisms of RNA-based silencing. This chapter describes a method for imaging fluorescently tagged Argonaute proteins in living mammalian cells using spinning disk confocal microscopy.

Hsp90 regulates the function of argonaute 2 and its recruitment to stress granules and P-bodies.

Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study, we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological inhibition of Hsp90 was associated with reduced microRNA- and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs, but interestingly, protein activator of the double-stranded RNA-activated protein kinase (PACT), another Dicer cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together, our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose that Ago2 and PACT form a complex that functions at the level of SGs.