Double-stranded RNA (dsRNA) produced during viral replication is believed to be the critical trigger for activation of antiviral immunity mediated by the RNA helicase enzymes retinoic acid–inducible gene I (RIG-I) and melanoma differentiation–associated gene 5 (MDA5). We showed that influenza A virus infection does not generate dsRNA and that RIG-I is activated by viral genomic single-stranded RNA (ssRNA) bearing 5′-phosphates. This is blocked by the influenza protein nonstructured protein 1 (NS1), which is found in a complex with RIG-I in infected cells. These results identify RIG-I as a ssRNA sensor and potential target of viral immune evasion and suggest that its ability to sense 5'-phosphorylated RNA evolved in the innate immune system as a means of discriminating between self and nonself.

Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.

The human coronaviruses (CoVs) severe acute respiratory syndrome (SARS)-CoV and NL63 employ angiotensin-converting enzyme 2 (ACE2) for cell entry. It was shown that recombinant SARS-CoV spike protein (SARS-S) downregulates ACE2 expression and thereby promotes lung injury. Whether NL63-S exerts a similar activity is yet unknown. We found that recombinant SARS-S bound to ACE2 and induced ACE2 shedding with higher efficiency than NL63-S. Shedding most likely accounted for the previously observed ACE2 downregulation but was dispensable for viral replication. Finally, SARS-CoV but not NL63 replicated efficiently in ACE2-positive Vero cells and reduced ACE2 expression, indicating robust receptor interference in the context of SARS-CoV but not NL63 infection.

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock. Broad-range anti-infective drugs are well known against bacteria, fungi, and parasites. These pathogens maintain their own metabolism distinctive from that of the host. Broad-range drugs can be obtained by targeting elements that several of these organisms have in common. In contrast, target overlap between different viruses is minimal. The replication of viruses is highly interweaved with the metabolism of the host cell. A high potential in the development of antivirals with broad activity might therefore reside in the identification of host factors elemental to virus replication. In this work we followed a systems biology approach, screening for interactions between virus and host proteins by employing an automated yeast-two-hybrid setup. Upon binding of a viral protein to cyclophilins the screen led to the identification of the Calcineurin/NFAT pathway possibly being involved in the pathogenesis of SARS-Coronavirus. Secondly, cyclophilins were suggested to play an elemental role in virus replication since cyclosporin A inhibited replication of all Coronavirus prototype members tested. This large range of viruses includes common cold viruses, the SARS agent, as well as a range of animal viruses. For the first time this work shows that an undirected, systems-biology approach could identify a host-encoded, broad-range antiviral target.