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.

Mutational signature dynamics indicate SARS-CoV-2's evolutionary capacity is driven by host antiviral molecules.

The COVID-19 pandemic has been characterised by sequential variant-specific waves shaped by viral, individual human and population factors. SARS-CoV-2 variants are defined by their unique combinations of mutations and there has been a clear adaptation to more efficient human infection since the emergence of this new human coronavirus in late 2019. Here, we use machine learning models to identify shared signatures, i.e., common underlying mutational processes and link these to the subset of mutations that define the variants of concern (VOCs). First, we examined the global SARS-CoV-2 genomes and associated metadata to determine how viral properties and public health measures have influenced the magnitude of waves, as measured by the number of infection cases, in different geographic locations using regression models. This analysis showed that, as expected, both public health measures and virus properties were associated with the waves of regional SARS-CoV-2 reported infection numbers and this impact varies geographically. We attribute this to intrinsic differences such as vaccine coverage, testing and sequencing capacity and the effectiveness of government stringency. To assess underlying evolutionary change, we used non-negative matrix factorisation and observed three distinct mutational signatures, unique in their substitution patterns and exposures from the SARS-CoV-2 genomes. Signatures 1, 2 and 3 were biased to C→T, T→C/A→G and G→T point mutations. We hypothesise assignments of these mutational signatures to the host antiviral molecules APOBEC, ADAR and ROS respectively. We observe a shift amidst the pandemic in relative mutational signature activity from predominantly Signature 1 changes to an increasingly high proportion of changes consistent with Signature 2. This could represent changes in how the virus and the host immune response interact and indicates how SARS-CoV-2 may continue to generate variation in the future. Linkage of the detected mutational signatures to the VOC-defining amino acids substitutions indicates the majority of SARS-CoV-2's evolutionary capacity is likely to be associated with the action of host antiviral molecules rather than virus replication errors.

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.