Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture.

Cyclophilin A (CypA) is an important host factor in the replication of a variety of RNA viruses. Also the replication of several nidoviruses was reported to depend on CypA, although possibly not to the same extent. These prior studies are difficult to compare, since different nidoviruses, cell lines and experimental set-ups were used. Here, we investigated the CypA dependence of three distantly related nidoviruses that can all replicate in Huh7 cells: the arterivirus equine arteritis virus (EAV), the alphacoronavirus human coronavirus 229E (HCoV-229E), and the betacoronavirus Middle East respiratory syndrome coronavirus (MERS-CoV). The replication of these viruses was compared in the same parental Huh7 cells and in CypA-knockout Huh7 cells generated using CRISPR/Cas9-technology. CypA depletion reduced EAV yields by ~ 3-log, whereas MERS-CoV progeny titers were modestly reduced (3-fold) and HCoV-229E replication was unchanged. This study reveals that the replication of nidoviruses can differ strikingly in its dependence on cellular CypA.

Alisporivir inhibits MERS- and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model.

Currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like SARS- and MERS-coronavirus. We here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin A-analog, inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. Nevertheless, our data provide a basis to further explore the potential of Cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication.

Antiviral Innate Immune Response Interferes with the Formation of Replication-Associated Membrane Structures Induced by a Positive-Strand RNA Virus.

Infection with nidoviruses like corona- and arteriviruses induces a reticulovesicular network of interconnected endoplasmic reticulum (ER)-derived double-membrane vesicles (DMVs) and other membrane structures. This network is thought to accommodate the viral replication machinery and protect it from innate immune detection. We hypothesized that the innate immune response has tools to counteract the formation of these virus-induced replication organelles in order to inhibit virus replication. Here we have investigated the effect of type I interferon (IFN) treatment on the formation of arterivirus-induced membrane structures. Our approach involved ectopic expression of arterivirus nonstructural proteins nsp2 and nsp3, which induce DMV formation in the absence of other viral triggers of the interferon response, such as replicating viral RNA. Thus, this setup can be used to identify immune effectors that specifically target the (formation of) virus-induced membrane structures. Using large-scale electron microscopy mosaic maps, we found that IFN-ß treatment significantly reduced the formation of the membrane structures. Strikingly, we also observed abundant stretches of double-membrane sheets (a proposed intermediate of DMV formation) in IFN-ß-treated samples, suggesting the disruption of DMV biogenesis. Three interferon-stimulated gene products, two of which have been reported to target the hepatitis C virus replication structures, were tested for their possible involvement, but none of them affected membrane structure formation. Our study reveals the existence of a previously unknown innate immune mechanism that antagonizes the viral hijacking of host membranes. It also provides a solid basis for further research into the poorly understood interactions between the innate immune system and virus-induced replication structures. IMPORTANCE: Viruses with a positive-strand RNA genome establish a membrane-associated replication organelle by hijacking and remodeling intracellular host membranes, a process deemed essential for their efficient replication. It is unknown whether the cellular innate immune system can detect and/or inhibit the formation of these membrane structures, which could be an effective mechanism to delay viral RNA replication. In this study, using an expression system that closely mimics the formation of arterivirus replication structures, we show for the first time that IFN-ß treatment clearly reduces the amount of induced membrane structures. Moreover, drastic morphological changes were observed among the remaining structures, suggesting that their biogenesis was impaired. Follow-up experiments suggested that host cells contain a hitherto unknown innate antiviral mechanism, which targets this common feature of positive-strand RNA virus replication. Our study provides a strong basis for further research into the interaction of the innate immune system with membranous viral replication organelles.

Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.

Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc-dependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-ß mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual-specificity proteases.

Arterivirus and nairovirus ovarian tumor domain-containing Deubiquitinases target activated RIG-I to control innate immune signaling.

The innate immune response constitutes the first line of defense against viral infection and is extensively regulated through ubiquitination. The removal of ubiquitin from innate immunity signaling factors by deubiquitinating enzymes (DUBs) therefore provides a potential opportunity for viruses to evade this host defense system. It was previously found that specific proteases encoded by the unrelated arteri- and nairoviruses resemble the ovarian tumor domain-containing (OTU) family of DUBs. In arteriviruses, this domain has been characterized before as a papain-like protease (PLP2) that is also involved in replicase polyprotein processing. In nairoviruses, the DUB resides in the polymerase protein but is not essential for RNA replication. Using both in vitro and cell-based assays, we now show that PLP2 DUB activity is conserved in all members of the arterivirus family and that both arteri- and nairovirus DUBs inhibit RIG-I-mediated innate immune signaling when overexpressed. The potential relevance of RIG-I-like receptor (RLR) signaling for the innate immune response against arterivirus infection is supported by our finding that in mouse embryonic fibroblasts, the production of beta interferon primarily depends on the recognition of arterivirus RNA by the pattern-recognition receptor MDA5. Interestingly, we also found that both arteri- and nairovirus DUBs inhibit RIG-I ubiquitination upon overexpression, suggesting that both MDA5 and RIG-I have a role in countering infection by arteriviruses. Taken together, our results support the hypothesis that arteri- and nairoviruses employ their deubiquitinating potential to inactivate cellular proteins involved in RLR-mediated innate immune signaling, as exemplified by the deubiquitination of RIG-I.

Development and RNA-synthesizing activity of coronavirus replication structures in the absence of protein synthesis.

The RNA replication and transcription complex of coronaviruses is associated with an elaborate reticulovesicular network (RVN) of modified endoplasmic reticulum. Using cycloheximide and puromycin, we have studied the effect of translation inhibition on the RNA synthesis of severe acute respiratory syndrome coronavirus and mouse hepatitis virus. Both inhibitors prevented the usual exponential increase in viral RNA synthesis, with immunofluorescence and electron microscopy indicating that RVN development came to a standstill. Nevertheless, limited RNA synthesis was supported, implying that continued translation is not an absolute requirement and suggesting a direct link between RVN formation and accumulation of coronavirus proteins.

The effect of Trim5 polymorphisms on the clinical course of HIV-1 infection.

The antiviral factor tripartite interaction motif 5alpha (Trim5alpha) restricts a broad range of retroviruses in a species-specific manner. Although human Trim5alpha is unable to block HIV-1 infection in human cells, a modest inhibition of HIV-1 replication has been reported. Recently two polymorphisms in the Trim5 gene (H43Y and R136Q) were shown to affect the antiviral activity of Trim5alpha in vitro. In this study, participants of the Amsterdam Cohort studies were screened for polymorphisms at amino acid residue 43 and 136 of the Trim5 gene, and the potential effects of these polymorphisms on the clinical course of HIV-1 infection were analyzed. In agreement with the reported decreased antiviral activity of Trim5alpha that contains a Y at amino acid residue 43 in vitro, an accelerated disease progression was observed for individuals who were homozygous for the 43Y genotype as compared to individuals who were heterozygous or homozygous for the 43H genotype. A protective effect of the 136Q genotype was observed but only after the emergence of CXCR4-using (X4) HIV-1 variants and when a viral load of 10(4.5) copies per ml plasma was used as an endpoint in survival analysis. Interestingly, naive CD4 T cells, which are selectively targeted by X4 HIV-1, revealed a significantly higher expression of Trim5alpha than memory CD4 T cells. In addition, we observed that the 136Q allele in combination with the -2GG genotype in the 5'UTR was associated with an accelerated disease progression. Thus, polymorphisms in the Trim5 gene may influence the clinical course of HIV-1 infection also underscoring the antiviral effect of Trim5alpha on HIV-1 in vivo.

The presence of the Trim5alpha escape mutation H87Q in the capsid of late stage HIV-1 variants is preceded by a prolonged asymptomatic infection phase.

BACKGROUND: Recently, the tripartite interaction motif 5alpha (Trim5alpha) has been identified as an inhibitory factor blocking infection of a broad range of retroviruses in a species-specific manner. In particular, HIV-1 replication can be efficiently blocked by Trim5alpha from Old World monkeys. The cyclophilin A binding region in the HIV-1 capsid is believed to be the viral determinant for Trim5alpha, and mutations in this region lift the restriction in simian cells. Human Trim5alpha is also able to inhibit HIV-1 replication in vitro, implying that Trim5alpha may contribute to host control of HIV-1 replication in vivo. METHODS: HIV-1 variants from participants of the Amsterdam cohort studies were analysed for Trim5alpha escape mutations in the capsid. Patients who harboured HIV-1 variants with Trim5alpha escape mutations were compared with patients who lacked such variants in terms of clinical course of infection. RESULTS: Trim5alpha escape mutants emerged in the late phase of infection and were ultimately present in 13.7% of HIV-1 infected individuals. Patients who developed Trim5alpha escape variants late in infection had a significantly lower set-point plasma viral RNA load and concomitantly a prolonged asymptomatic survival as compared to individuals who lacked Trim5alpha escape mutants. This protective effect was stronger in individuals who later developed X4 variants. In addition, X4-emergence was delayed in individuals who later developed Trim5alpha escape variants, compatible with suppression of viral replication. CONCLUSION: Our data are compatible with Trim5alpha-mediated suppression of viral replication, resulting in prolonged asymptomatic survival and ultimately the selection of Trim5alpha escape variants.