The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

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

Flavivirus RNA methylation

The 5′ end of eukaryotic mRNA contains the type-1 (m7GpppNm) or type-2 (m7GpppNmNm) cap structure. Many viruses have evolved various mechanisms to develop their own capping enzymes (e.g. flavivirus and coronavirus) or to 'steal' caps from host mRNAs (e.g. influenza virus). Other viruses have developed 'cap-mimicking' mechanisms by attaching a peptide to the 5′ end of viral RNA (e.g. picornavirus and calicivirus) or by having a complex 5′ RNA structure (internal ribosome entry site) for translation initiation (e.g. picornavirus, pestivirus and hepacivirus). Here we review the diverse viral RNA capping mechanisms. Using flavivirus as a model, we summarize how a single methyltransferase catalyses two distinct N-7 and 2′-O methylations of viral RNA cap in a sequential manner. For antiviral development, a structural feature unique to the flavivirus methyltransferase was successfully used to design selective inhibitors that block viral methyltransferase without affecting host methyltransferases. Functionally, capping is essential for prevention of triphosphate-triggered innate immune activation; N-7 methylation is critical for enhancement of viral translation; and 2′-O methylation is important for subversion of innate immune response during viral infection. Flaviviruses defective in 2′-O methyltransferase are replicative, but their viral RNAs lack 2′-O methylation and are recognized and eliminated by the host immune response. Such mutant viruses could be rationally designed as live attenuated vaccines. This concept has recently been proved with Japanese encephalitis virus and dengue virus. The findings obtained with flavivirus should be applicable to other RNA viruses. © 2014 SGM

Rational design of a flavivirus vaccine through abolishing viral RNA 2′-O methylation

Viruses that replicate in the cytoplasm cannot access to the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2’-O cap of their RNA; alternatively, they ‘snatch’ host mRNA cap to form the 5’-end of viral RNA. The function of 2’-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2’-O methylation is replicative; but its viral RNA lacks 2’-O methylation, and is recognized and subsequently eliminated by host immune response. Such mutant virus could be rationally designed as a live attenuated vaccine. Here we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus pathogen, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2’-O methylation was stable in cell culture after continuous passaging for >30 days. The mutant virus was attenuated in mice, and elicited robust humoral and cellular immune response. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2’-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and non-flaviviruses that encode their own viral 2’-O methyltransferases

Plasmablasts During Acute Dengue Infection Represent a Small Subset of a Broader Virus-specific Memory B Cell Pool

Dengue is endemic in tropical countries worldwide and the four dengue virus serotypes often co-circulate. Infection with one serotype results in high titers of cross-reactive antibodies produced by plasmablasts, protecting temporarily against all serotypes, but impairing protective immunity in subsequent infections. To understand the development of these plasmablasts, we analyzed virus-specific B cell properties in patients during acute disease and at convalescence. Plasmablasts were unrelated to classical memory cells expanding in the blood during early recovery. We propose that only a small subset of memory B cells is activated as plasmablasts during repeat infection and that plasmablast responses are not representative of the memory B cell repertoire after dengue infection