N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition.

The ongoing coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is associated with substantial morbidity and mortality. Understanding the immunological and pathological processes of coronavirus diseases is crucial for the rational design of effective vaccines and therapies for COVID-19. Previous studies showed that 2'-O-methylation of the viral RNA cap structure is required to prevent the recognition of viral RNAs by intracellular innate sensors. Here, we demonstrate that the guanine N7-methylation of the 5' cap mediated by coronavirus nonstructural protein 14 (nsp14) contributes to viral evasion of the type I interferon (IFN-I)-mediated immune response and pathogenesis in mice. A Y414A substitution in nsp14 of the coronavirus mouse hepatitis virus (MHV) significantly decreased N7-methyltransferase activity and reduced guanine N7-methylation of the 5' cap in vitro. Infection of myeloid cells with recombinant MHV harboring the nsp14-Y414A mutation (rMHVnsp14-Y414A) resulted in upregulated expression of IFN-I and ISG15 mainly via MDA5 signaling and in reduced viral replication compared to that of wild-type rMHV. rMHVnsp14-Y414A replicated to lower titers in livers and brains and exhibited an attenuated phenotype in mice. This attenuated phenotype was IFN-I dependent because the virulence of the rMHVnsp14-Y414A mutant was restored in Ifnar-/- mice. We further found that the comparable mutation (Y420A) in SARS-CoV-2 nsp14 (rSARS-CoV-2nsp14-Y420A) also significantly decreased N7-methyltransferase activity in vitro, and the mutant virus was attenuated in K18-human ACE2 transgenic mice. Moreover, infection with rSARS-CoV-2nsp14-Y420A conferred complete protection against subsequent and otherwise lethal SARS-CoV-2 infection in mice, indicating the vaccine potential of this mutant. IMPORTANCE Coronaviruses (CoVs), including SARS-CoV-2, the cause of COVID-19, use several strategies to evade the host innate immune responses. While the cap structure of RNA, including CoV RNA, is important for translation, previous studies indicate that the cap also contributes to viral evasion from the host immune response. In this study, we demonstrate that the N7-methylated cap structure of CoV RNA is pivotal for virus immunoevasion. Using recombinant MHV and SARS-CoV-2 encoding an inactive N7-methyltransferase, we demonstrate that these mutant viruses are highly attenuated in vivo and that attenuation is apparent at very early times after infection. Virulence is restored in mice lacking interferon signaling. Further, we show that infection with virus defective in N7-methylation protects mice from lethal SARS-CoV-2, suggesting that the N7-methylase might be a useful target in drug and vaccine development.

Physiologic RNA targets and refined sequence specificity of coronavirus EndoU.

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3' side of pyrimidines with a strong preference for U ↓ A and C ↓ A sequences (endoY ↓ A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5' NTR to the 3' NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2'-5' phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U ↓ A and C ↓ A sequences (endoY ↓ A) within viral (+) strand RNA to evade dsRNA-activated host responses.

APOBEC3-mediated restriction of RNA virus replication.

APOBEC3 family members are cytidine deaminases with roles in intrinsic responses to infection by retroviruses and retrotransposons, and in the control of other DNA viruses, such as herpesviruses, parvoviruses and hepatitis B virus. Although effects of APOBEC3 members on viral DNA have been demonstrated, it is not known whether they edit RNA genomes through cytidine deamination. Here, we investigated APOBEC3-mediated restriction of Coronaviridae. In experiments in vitro, three human APOBEC3 proteins (A3C, A3F and A3H) inhibited HCoV-NL63 infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. APOBEC3-mediated restriction was partially dependent on enzyme activity, and was reduced by the use of enzymatically inactive APOBEC3. Moreover, APOBEC3 proteins bound to the coronaviral nucleoprotein, and this interaction also affected viral replication. Although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of APOBEC-mediated restriction of RNA virus infections.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

SARS-CoV and IFN: Too Little, Too Late.

Dysregulated type I interferon (IFN-I) expression can lead to severe pathology and disease. In this issue of Cell Host & Microbe, Channappanavar et al. (2016) use a SARS-coronavirus animal model to describe how rapid and robust virus replication with delayed IFN-I can lead to lung immunopathology, with fatal outcomes.

Murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis.

UNLABELLED: Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE: Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

To sense or not to sense viral RNA--essentials of coronavirus innate immune evasion.

An essential function of innate immunity is to distinguish self from non-self and receptors have evolved to specifically recognize viral components and initiate the expression of antiviral proteins to restrict viral replication. Coronaviruses are RNA viruses that replicate in the host cytoplasm and evade innate immune sensing in most cell types, either passively by hiding their viral signatures and limiting exposure to sensors or actively, by encoding viral antagonists to counteract the effects of interferons. Since many cytoplasmic viruses exploit similar mechanisms of innate immune evasion, mechanistic insight into the direct interplay between viral RNA, viral RNA-processing enzymes, cellular sensors and antiviral proteins will be highly relevant to develop novel antiviral targets and to restrict important animal and human infections.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Sequestration by IFIT1 impairs translation of 2'O-unmethylated capped RNA.

Viruses that generate capped RNA lacking 2'O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2'O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2'O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2'O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2'O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells.

Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential.

The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

Analysis of rotavirus species diversity and evolution including the newly determined full-length genome sequences of rotavirus F and G.

Rotaviruses are a leading cause of viral acute gastroenteritis in humans and animals. Eight different rotavirus species (A-H) have been defined based on antigenicity and nucleotide sequence identities of the VP6 gene. Here, the first complete genome sequences of rotavirus F (strain 03V0568) and G (strain 03V0567) with lengths of 18,341 and 18,186bp, respectively, are described. Both viruses have open reading frames for rotavirus proteins VP1 to VP7 and NSP1 to NSP5 located at the 11 genome segments. Nucleotide sequence identities to other rotaviruses ranged between 29.8% (NSP1 gene) and 61.7% (VP1 gene) for rotavirus F and between 29.3% (NSP1-2 gene) and 65.9% (NSP2 gene) for rotavirus G, thus confirming their classification as separate virus species. Encoded proteins revealed remarkable sequence differences among the rotavirus species. In contrast, the non-coding 5'-terminal sequences of the genome segments are highly conserved among all rotavirus species. Different 3'-terminal consensus sequences are found between rotavirus A/D/F, rotavirus C and rotavirus B/G/H. Phylogenetic analyses indicated a separation of rotaviruses in two major clades consisting of rotavirus A/C/D/F and rotavirus B/G/H. Within these clades, rotavirus F mainly clustered with rotavirus D and rotavirus G with rotavirus B. In addition, differentiation among mammalian and avian rotavirus A strains, host-specific evolution of rotavirus B and C as well as an ancient reassortment event between avian rotavirus A and D are indicated by the phylogenetic data. These results underline the high diversity of rotaviruses as a result of a complex evolutionary history.

Deep phylogeographic divergence and cytonuclear discordance in the grasshopper Oedaleus decorus.

The grasshopper Oedaleus decorus is a thermophilic insect with a large, mostly south-Palaearctic distribution range, stretching from the Mediterranean regions in Europe to Central-Asia and China. In this study, we analyzed the extent of phylogenetic divergence and the recent evolutionary history of the species based on 274 specimens from 26 localities across the distribution range in Europe. Phylogenetic relationships were determined using sequences of two mitochondrial loci (ctr, ND2) with neighbour-joining and Bayesian methods. Additionally, genetic differentiation was analyzed based on mitochondrial DNA and 11 microsatellite markers using F-statistics, model-free multivariate and model-based Bayesian clustering approaches. Phylogenetic analyses detected consistently two highly divergent, allopatrically distributed lineages within O. decorus. The divergence among these Western and Eastern lineages meeting in the region of the Alps was similar to the divergence of each lineage to the sister species O. asiaticus. Genetic differentiation for ctr was extremely high between Western and Eastern grasshopper populations (F(ct)=0.95). Microsatellite markers detected much lower but nevertheless very significant genetic structure among population samples. The nuclear data also demonstrated a case of cytonuclear discordance because the affiliation with mitochondrial lineages was incongruent in Northern Italy. Taken together these results provide evidence of an ancient separation within Oedaleus and either historical introgression of mtDNA among lineages and/or ongoing sex-specific gene flow in this grasshopper. Our study stresses the importance of multilocus approaches for unravelling the history and status of taxa of uncertain evolutionary divergence.

Rat hepatitis E virus: geographical clustering within Germany and serological detection in wild Norway rats (Rattus norvegicus).

Zoonotic hepatitis E virus (HEV) infection in industrialised countries is thought to be caused by transmission from wild boar, domestic pig and deer as reservoir hosts. The detection of HEV-specific antibodies in rats and other rodents has suggested that these animals may represent an additional source for HEV transmission to human. Recently, a novel HEV (ratHEV) was detected in Norway rats from Hamburg, Germany, showing the typical genome organisation but a high nucleotide and amino acid sequence divergence to other mammalian and to avian HEV strains. Here we describe the multiple detection of ratHEV RNA and HEV-specific antibodies in Norway rats from additional cities in north-east and south-west Germany. The complete genome analysis of two novel strains from Berlin and Stuttgart confirmed the association of ratHEV to Norway rats. The present data indicated a continuing existence of this virus in the rat populations from Berlin and Hamburg. The phylogenetic analysis of a short segment of the open reading frame 1 confirmed a geographical clustering of the corresponding sequences. Serological investigations using recombinant ratHEV and genotype 3 capsid protein derivatives demonstrated antigenic differences which might be caused by the high amino acid sequence divergence in the immunodominant region. The high amount of animals showing exclusively ratHEV RNA or anti-ratHEV antibodies suggested a non-persistent infection in the Norway rat. Future studies have to prove the transmission routes of the virus in rat populations and its zoonotic potential. The recombinant ratHEV antigen generated here will allow future seroepidemiological studies to differentiate ratHEV and genotype 3 infections in humans and animals.

Tula virus infections in the Eurasian water vole in Central Europe.

Recent reports of novel hantaviruses in shrews and moles and the detection of rodent-borne hantaviruses in different rodent species raise important questions about their host range and specificity, evolution, and host adaptation. Tula virus (TULV), a European hantavirus, is believed to be slightly or non-pathogenic in humans and was initially detected in the common vole Microtus arvalis, the East European vole M. levis (formerly rossiaemeridionalis), and subsequently in other Microtus species. Here we report the first multiple RT-PCR detection and sequence analyses of TULV in the Eurasian water vole Arvicola amphibius from different regions in Germany and Switzerland. Additional novel TULV S-, M-, and L-segment sequences were obtained from M. arvalis and M. agrestis trapped in Germany at sites close to trapping sites of TULV-RT-PCR-positive water voles. Serological investigations using a recombinant TULV nucleocapsid protein revealed the presence of TULV-reactive antibodies in RT-PCR-positive and a few RT-PCR-negative water voles. Phylogenetic analyses revealed a geographical clustering of the novel S-, M-, and L-segment sequences from A. amphibius with those of M. arvalis- and M. agrestis-derived TULV lineages, and may suggest multiple TULV spillover or a potential host switch to A. amphibius. Future longitudinal studies of sympatric Microtus and Arvicola populations and experimental infection studies have to prove the potential of A. amphibius as an additional TULV reservoir host.

Phylogenetic analysis of Puumala virus subtype Bavaria, characterization and diagnostic use of its recombinant nucleocapsid protein.

Puumala virus (PUUV) is the predominant hantavirus species in Germany causing large numbers of mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS). During an outbreak in South-East Germany in 2004 a novel PUUV subtype designated Bavaria was identified as the causative agent of HFRS in humans [1]. Here we present a molecular characterization of this PUUV strain by investigating novel partial and almost entire nucleocapsid (N) protein-encoding small (S-) segment sequences and partial medium (M-) segment sequences from bank voles (Myodes glareolus) trapped in Lower Bavaria during 2004 and 2005. Phylogenetic analyses confirmed their classification as subtype Bavaria, which is further subdivided into four geographical clusters. The entire N protein, harbouring an amino-terminal hexahistidine tag, of the Bavarian strain was produced in yeast Saccharomyces cerevisiae and showed a slightly different reactivity with N-specific monoclonal antibodies, compared to the yeast-expressed N protein of the PUUV strain Vranica/Hällnäs. Endpoint titration of human sera from different parts of Germany and from Finland revealed only very slight differences in the diagnostic value of the different recombinant proteins. Based on the novel N antigen indirect and monoclonal antibody capture IgG-ELISAs were established. By using serum panels from Germany and Finland their validation demonstrated a high sensitivity and specificity. In summary, our investigations demonstrated the Bavarian PUUV strain to be genetically divergent from other PUUV strains and the potential of its N protein for diagnostic applications.

Novel hepatitis E virus genotype in Norway rats, Germany.

Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E-like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus.