The positive-strand RNA viruses initiate their amplification in the cell from a single genome delivered by virion. This single RNA molecule needs to become involved in replication process before it is recognized and degraded by cellular machinery. In this study, we show that distantly related New World and Old World alphaviruses have independently evolved to utilize different cellular stress granule-related proteins for assembly of complexes, which recruit viral genomic RNA and facilitate formation of viral replication complexes (vRCs). Venezuelan equine encephalitis virus (VEEV) utilizes all members of the Fragile X syndrome (FXR) family, while chikungunya and Sindbis viruses exploit both members of the G3BP family. Despite being in different families, these proteins share common characteristics, which determine their role in alphavirus replication, namely, the abilities for RNA-binding and for self-assembly into large structures. Both FXR and G3BP proteins interact with virus-specific, repeating amino acid sequences located in the C-termini of hypervariable, intrinsically disordered domains (HVDs) of viral nonstructural protein nsP3. We demonstrate that these host factors orchestrate assembly of vRCs and play key roles in RNA and virus replication. Only knockout of all of the homologs results in either pronounced or complete inhibition of replication of different alphaviruses. The use of multiple homologous proteins with redundant functions mediates highly efficient recruitment of viral RNA into the replication process. This independently evolved acquisition of different families of cellular proteins by the disordered protein fragment to support alphavirus replication suggests that other RNA viruses may utilize a similar mechanism of host factor recruitment for vRC assembly. The use of different host factors by alphavirus species may be one of the important determinants of their pathogenesis.

The Old World alphaviruses are emerging human pathogens with an ability to cause widespread epidemics. The latest epidemic of Chikungunya virus, from 2005 to 2007, affected over 40 countries in Africa, Asia, and Europe. The Old World alphaviruses are highly cytopathic and known to evade the cellular antiviral response by inducing global inhibition of transcription in vertebrate cells. This function was shown to be mediated by their nonstructural nsP2 protein; however, the detailed mechanism of this phenomenon has remained unknown. Here, we report that nsP2 proteins of Sindbis, Semliki Forest, and Chikungunya viruses inhibit cellular transcription by inducing rapid degradation of Rpb1, a catalytic subunit of the RNAPII complex. This degradation of Rpb1 is independent of the nsP2-associated protease activity, but, instead, it proceeds through nsP2-mediated Rpb1 ubiquitination. This function of nsP2 depends on the integrity of the helicase and S-adenosylmethionine (SAM)-dependent methyltransferase-like domains, and point mutations in either of these domains abolish Rpb1 degradation. We go on to show that complete degradation of Rpb1 in alphavirus-infected cells occurs within 6 h postinfection, before other previously described virus-induced changes in cell physiology, such as apoptosis, autophagy, and inhibition of STAT1 phosphorylation, are detected. Since Rpb1 is a subunit that catalyzes the polymerase reaction during RNA transcription, degradation of Rpb1 plays an indispensable role in blocking the activation of cellular genes and downregulating cellular antiviral response. This indicates that the nsP2-induced degradation of Rpb1 is a critical mechanism utilized by the Old World alphaviruses to subvert the cellular antiviral response. E fficient replication of viruses in infected hosts relies not only on their replicative machinery and expression of virus-specific nonstructural and structural proteins but also on their ability to interfere with the antiviral response mounted in cells of both vertebrate and invertebrate origin. Different viruses have evolved numerous mechanisms to inhibit the cellular antiviral response. While the vast majority of them interfere with specific steps of cellular signaling pathways, the highly cytopathic alphaviruses induce a global inhibition of cellular transcription (15). The entire alphavirus life cycle proceeds in the cytoplasm and is completed within 24 to 48 h postinfection (p.i.) (33). Therefore, functional nuclei appear to play no role in alphavirus replication. This allows global inhibition of cellular transcription to serve as an efficient means of inhibiting the antiviral response without affecting virus replication and egress. The inability of infected cells to activate cytokine and chemokine expression makes them incapable of signaling and, importantly, of releasing type I interferon (IFN), which could induce an antiviral state in as-yet-uninfected cells.The alphavirus genome is a single-stranded RNA of positive polarity with a cap at the 5= terminus and poly(A) at the...

Alphaviruses are a group of single-stranded RNA viruses with genomes of positive polarity. They are divided into two geographically isolated groups: the Old World and the New World alphaviruses. Despite their similar genome organizations and virion structures, they differ in many aspects of pathogenesis and interaction with the host cell. Here we present new data highlighting previously unknown differences between these two groups. We found that nsP3 proteins of Sindbis virus (SINV) and Venezuelan equine encephalitis virus (VEEV) form cytoplasmic complexes with different morphologies and protein compositions. Unlike the amorphous aggregates formed by SINV nsP3 and other Old World alphavirus-specific nsP3s, VEEV nsP3 forms unique, large spherical structures with striking symmetry. Moreover, VEEV nsP3 does not interact with proteins previously identified as major components of SINV nsP3 complexes, such as G3BP1 and G3BP2. Importantly, the morphology of the complexes and the specificity of the interaction with cellular proteins are largely determined by the hypervariable domain (HVD) of nsP3. Replacement of the VEEV nsP3 HVD with the corresponding domain of SINV nsP3 rendered this protein capable of interaction with G3BPs. Conversely, replacement of the SINV nsP3 HVD with that of VEEV abolished SINV nsP3's interaction with G3BPs. The replacement of natural HVDs with those from heterologous viruses did not abrogate virus replication, despite these fragments demonstrating very low levels of sequence identity. Our data suggest that in spite of the differences in morphology and composition of the SINV-and VEEV-specific nsP3 complexes, it is likely that they have similar functions in virus replication and modification of the cellular environment.

The transmembrane serine protease TMPRSS2 is indispensable for S protein priming of the MERS, SARS-CoV, and SARS-CoV2 coronaviruses, a process that is necessary for entry of the virus into host cells. Therefore, inhibiting TMPRSS2 holds promise as an approach toward preventing transmission of coronaviruses. Herein, we developed an in vitro system to measure TMPRSS2 activity and tested the inhibition of TMPRSS2 by several synthetic and natural protease inhibitors. Camostat mesylate and bromhexine hydrochloride (BHH) inhibited TMPRSS2 proteolytic function. In addition, we identified the small molecule 4-(2-aminomethyl)benzenesulfonyl fluoride (AEBSF) and the human, anti-inflammatory protein alpha 1 antitrypsin (A1AT) as inhibitors of TMPRSS2. AEBSF and A1AT inhibited TMPRSS2 activity in a dose-dependent manner. AEBSF and A1AT inhibited TMPRSS2 in the same range of concentrations (100-0.1 μM). We suggest that treatment with these inhibitors, particularly A1AT, which is an FDA-approved drug, might be effective in limiting SARS-CoV and SARS-CoV2 transmissibility and as a COVID-19 treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a ‘cytokine storm.’ In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.