Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

Interactions between Influenza A Virus Nucleoprotein and Gene Segment Untranslated Regions Facilitate Selective Modulation of Viral Gene Expression.

The influenza A virus (IAV) genome is divided into eight negative-sense, single-stranded RNA segments. Each segment exhibits a unique level and temporal pattern of expression; however, the exact mechanisms underlying the patterns of individual gene segment expression are poorly understood. We previously demonstrated that a single substitution in the viral nucleoprotein (NP:F346S) selectively modulates neuraminidase (NA) gene segment expression while leaving other segments largely unaffected. Given what is currently known about NP function, there is no obvious explanation for how changes in NP can selectively modulate the replication of individual gene segments. In this study, we found that the specificity of this effect for the NA segment is virus strain specific and depends on the untranslated region (UTR) sequences of the NA segment. While the NP:F346S substitution did not significantly alter the RNA binding or oligomerization activities of NP in vitro, it specifically decreased the ability of NP to promote NA segment viral RNA (vRNA) synthesis. In addition to NP residue F346, we identified two other adjacent aromatic residues in NP (Y385 and F479) capable of similarly regulating NA gene segment expression, suggesting a larger role for this domain in gene-segment specific regulation. Our findings reveal a novel role for NP in selective regulation of viral gene segment replication and provide a framework for understanding how the expression patterns of individual viral gene segments can be modulated during adaptation to new host environments. IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen that remains a significant source of morbidity and mortality. Escape from host immunity or emergence into new host species often requires mutations that modulate the functional activities of the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA), which are responsible for virus attachment to and release from host cells, respectively. Maintaining the functional balance between the activities of HA and NA is required for fitness across multiple host systems. Thus, selective modulation of viral gene expression patterns may be a key determinant of viral immune escape and cross-species transmission potential. We identified a novel mechanism by which the viral nucleoprotein (NP) gene can selectively modulate NA segment replication and gene expression through interactions with the segment UTRs. Our work highlights an unexpected role for NP in selective regulation of expression from the individual IAV gene segments.

A nano-luciferase expressing human coronavirus OC43 for countermeasure development.

The genetic diversity of the coronavirus (CoV) family poses a significant challenge for drug discovery and development. Traditional antiviral drugs often target specific viral proteins from specific viruses which limits their use, especially against novel emerging viruses. Antivirals with broad-spectrum activity overcome this limitation by targeting highly conserved regions or catalytic domains within viral proteins that are essential for replication. For rapid identification of small molecules with broad antiviral activity, assays with viruses representing family-wide genetic diversity are needed. Viruses engineered to express a reporter gene (i.e. luminescence, fluorescence, etc.) can increase the efficiency, sensitivity or precision of drug screening over classical measures of replication like observation of cytopathic effect or measurement of infectious titers. We have previously developed reporter virus systems for multiple other endemic, pandemic, epidemic and enzootic CoV. Human CoV OC43 (HCoV-OC43) is a human endemic CoV that causes respiratory infection with age-related exacerbations of pathogenesis. Here, we describe the development of a novel recombinant HCoV-OC43 reporter virus that expresses nano-luciferase (HCoV-OC43 nLuc), and its potential application for screening of antivirals against CoV.

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes as they. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

The oral nucleoside prodrug GS-5245 is efficacious against SARS-CoV-2 and other endemic, epidemic, and enzootic coronaviruses.

Despite the wide availability of several safe and effective vaccines that prevent severe COVID-19, the persistent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that can evade vaccine-elicited immunity remains a global health concern. In addition, the emergence of SARS-CoV-2 VOCs that can evade therapeutic monoclonal antibodies underscores the need for additional, variant-resistant treatment strategies. Here, we characterize the antiviral activity of GS-5245, obeldesivir (ODV), an oral prodrug of the parent nucleoside GS-441524, which targets the highly conserved viral RNA-dependent RNA polymerase (RdRp). We show that GS-5245 is broadly potent in vitro against alphacoronavirus HCoV-NL63, SARS-CoV, SARS-CoV-related bat-CoV RsSHC014, Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 WA/1, and the highly transmissible SARS-CoV-2 BA.1 Omicron variant. Moreover, in mouse models of SARS-CoV, SARS-CoV-2 (WA/1 and Omicron B1.1.529), MERS-CoV, and bat-CoV RsSHC014 pathogenesis, we observed a dose-dependent reduction in viral replication, body weight loss, acute lung injury, and pulmonary function with GS-5245 therapy. Last, we demonstrate that a combination of GS-5245 and main protease (Mpro) inhibitor nirmatrelvir improved outcomes in vivo against SARS-CoV-2 compared with the single agents. Together, our data support the clinical evaluation of GS-5245 against coronaviruses that cause or have the potential to cause human disease.

Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase.

Neuraminidase (NA) of human influenza H3N2 virus has evolved rapidly and been accumulating mutations for more than half-century. However, biophysical constraints that govern the evolutionary trajectories of NA remain largely elusive. Here, we show that among 70 natural mutations that are present in the NA of a recent human H3N2 strain, >10% are deleterious for an ancestral strain. By mapping the permissive mutations using combinatorial mutagenesis and next-generation sequencing, an extensive epistatic network is revealed. Biophysical and structural analyses further demonstrate that certain epistatic interactions can be explained by non-additive stability effect, which in turn modulates membrane trafficking and enzymatic activity of NA. Additionally, our results suggest that other biophysical mechanisms also contribute to epistasis in NA evolution. Overall, these findings not only provide mechanistic insights into the evolution of human influenza NA and elucidate its sequence-structure-function relationship, but also have important implications for the development of next-generation influenza vaccines.

The parts are greater than the whole: the role of semi-infectious particles in influenza A virus biology.

The influenza A virus (IAV) genome is incorporated into newly produced virions through a tightly orchestrated process that is one of the best studied examples of genome packaging by a segmented virus. Despite the remarkable selectivity and efficiency of this process, it is clear that the vast majority of IAV virions are unable to express the full set of essential viral gene products and are thus incapable of productive replication in the absence of complementation. Here, we attempt to reconcile the widespread production of these semi-infectious particles (SIPs) with the high efficiency and selectivity of IAV genome packaging. We also cover what is known and what remains unknown about the consequences of SIP production for the replication and evolution of viral populations.