It is shown that non-structural protein-15 (NSP15), a nuclease encoded by coronaviruses, can drive the acquisition of a class of insertion mutations, and numerous examples of potential furin cleavage site acquisition and replacement through insertion mutation during the normal course of coronavirus replication are found.
Abstract
Genetic variation in viruses is well known to arise from polymerase-driven nucleotide misincorporation. However, insertion and deletion (indel) mutations that occur at lower, largely unknown, frequencies can underly more dramatic phenotypic changes that emerge when advantageous. Using a human coronavirus (HCoV-OC43) construct that reports rare indel mutations, we show that non-structural protein-15 (NSP15), a nuclease encoded by coronaviruses, can drive the acquisition of a class of insertion mutations. Ultra-deep sequencing of both HCoV-OC43 and SARS-CoV-2 populations reveals a similar requirement for NSP15 during insertion mutant generation. Overall, the insertional mutation frequency exceeded 10-3/genome in these two coronaviruses. Analysis of thousands of HCoV-OC43 and SARS-CoV-2 insertion mutants reveals a mutational process in which NSP15 cuts viral RNA, yielding oligonucleotides that correspond to inserts that are acquired at distal genomic locations. The presence of an insertion mutation at the S1/S2 junction in the SARS-CoV-2 spike protein that generates a furin cleavage site and enhances viral transmissibility, may have been necessary for enabling the COVID19 pandemic. We found numerous examples of potential furin cleavage site acquisition and replacement through insertion mutation during the normal course of coronavirus replication. Such events are, therefore, likely commonplace in coronavirus populations of a size that occurs in nature.
Bovine viral diarrhea virus (BVDV) is a pathogen of globally significance in cattle that has two biotypes: non-cytopathogenic (non-cp) and cytopathogenic (cp). The cp biotype arises from the non-cp through genome rearrangements, which frequently involve the insertion of host cellular RNA sequences, often accompanied by viral genome duplications. Here, we performed a systematic analysis of insertions across all complete BVDV genomes available in GenBank. Despite a 10-fold increase in available sequences over the past 10 years (from 59 to 670), the repertoire of known rearrangements associated with the cp phenotype has expanded only modestly, with insertions occurring predominantly at five conserved genomic hotspots. Notably, independent acquisitions of similar insertions—such as DNAJC14 (Jiv), ubiquitin-like sequences, the NS4B-NS5A cleavage site, and the PYPDPQTLG motif—in phylogenetically unrelated virus lineages reflect a limited number of permissive sites. Our analysis further demonstrates that many insertions are mosaic and suggests multiple recombination events that are poorly compatible with template switching by viral polymerase. Mechanistically, we propose that non-replicative recombination provides the most consistent explanation for the observed insertion patterns, particularly common coupling of cellular RNA acquisitions with viral sequence duplications. Both the steric proximity of virus replication to the endoplasmic reticulum and the RNA end requirements (2′-3′ cyclic phosphate and 5′-OH) suggest that non-replicative recombination is likely mediated by the endoplasmic reticulum-associated IRE1 RNase and RtcB ligase, which are responsible for the alternative cytoplasmic splicing of cellular mRNA.
F. Perelygin, Y. Aleshina, Ekaterina Chistiakova et al.· Viruses· 0 citations
The SARS-CoV-2 nucleocapsid (N) protein is one of the most highly expressed proteins during infection and plays crucial roles in the protection and packaging of viral RNA, replication, suppression of the immune response and virus assembly. The N gene and the overlapping accessory open reading frame ORF9b have continually evolved throughout the circulation of the virus, likely due to ongoing adaptation to the human host. This genetic variability influences the interplay of the N protein and its interactors. Yet, little is known about how specific mutations within the N/ORF9b locus of Variants of Concern (VOCs) shape the progression and outcome of infection. Here, we use a multi-omics approach to decipher how these genetic alterations reprogram the host cell by creating Wuhan-Hu-1-based recombinant viruses carrying an isogenic backbone with respective N mutations from the VOCs (called rNs) and comparing their effects at the transcriptome and proteome levels. We found that the mutations induce distinct transcriptional and translational alterations: rN-Alpha drives a stealth-like infection characterized by sustained translation efficiency and specific evasion of the 2’-5’-oligoadenylate synthetase (OAS) innate immune sensor, whereas rN-Delta and rN-BA.2 trigger a highly elevated inflammatory response. For rN-Delta, hyperphosphorylation of the N-protein drives cellular stress culminating in necroptotic cell death. Because these viruses differ only within the N/ORF9b locus, N sequence variation emerges as a determinant of infection outcome in its own right, warranting increased surveillance attention.
J. Berger, Jonas Schröder, Timothy K. Soh et al.· bioRxiv· 0 citations
Comparative genomic studies of Marek’s disease virus (MDV) conducted in the past two decades have proven powerful for identifying genomic loci driving historical shifts in virulence. However, past efforts were limited to comparing a few strains at a time, lacked access to certain regions of the MDV genome, and were restricted in their ability to characterize viral phenotypes. To address these limitations, we performed whole-genome sequencing on a collection of 65 MDV strains previously characterized using a standardized phenotyping assay. In addition to phylogenetic and recombination analyses, this approach enabled us to perform a genome-wide association study of virulence for this pathogen. In total, we identified 10 genome-wide significant loci associated with virulence, including a tandem repeat variant resulting from two alternative versions of a 132-bp repeating motif. Our findings support prior descriptions of virulence as a complex trait in MDV, and highlight the potential contribution of repeat-based and intergenic variants to the phenotypic diversity of herpesviruses.
Alejandro Ortigas-Vásquez, Utsav Pandey, A. Bell et al.· Science Advances· 0 citations
HIV transmission from one individual to another occurs by one or a small number of virions followed by spread and genetic diversification into a complex quasispecies. To understand the early events in this process, we investigated how HIV-1 genomes diversify within the first two to three weeks after transmission by use of ultra-deep single subgenomic sequencing of over 10,000 plasma RNA genomes in each of a cohort of 15 individuals in acute infection. This approach confirmed transmission of one or a few transmitted/founder (TF) viral lineages and very limited early divergence from the founder sequences. Most observed variants that differed from the TF included single nucleotide changes attributable to HIV-1 reverse transcriptase (RT) error or host APOBEC3G/F activity. Comparing the number of expected versus observed changes after transmission indicated that most de novo mutations do not persist in the virus population, consistent with strong purifying selection. We found little evidence that early diversification is driven by reversions to subtype consensus or by cytotoxic T lymphocyte pressure, although rare multi-mutation lineages suggest occasional influences. Together, these findings indicate that early HIV-1 evolution is influenced by stochastic and host-mediated mutational processes (e.g., APOBEC3G/F) filtered by strong purifying selection. The strong purifying selection observed in the early weeks of HIV-1 infection may provide an opportunity to investigate the potential of new interventions to induce viremic control, such as combinations of broadly neutralizing antibodies, cellular immunotherapy, or mRNA therapeutic vaccination. AUTHOR SUMMARY When a person acquires HIV, especially through sexual transmission, infection is usually established by just one or a few viral variants. However, over the early months and years of infection, these viral variants accumulate mutations until almost no two viral genomes are identical in a typical sample. Here, we sequenced tens of thousands of viral variants in the early weeks after transmission to understand the early events that contribute to this vast viral diversification. We found that the accumulation of mutations was slower than expected, implying a selection against HIV-1 diversification in acute infection, potentially leaving a window of low genetic diversity for the study of new interventions towards inducing viremic control, such as immunotherapy or mRNA vaccination. Of the early viral mutations that were observed, many were induced by host enzymes, rather than from errors by the viral enzyme used for replication. Our results provide more context for understanding HIV evolution and provide a deep sampling of viral diversity after transmission.
A. Capoferri, V. Boltz, W. Shao et al.· bioRxiv· 0 citations
The internal ribosome entry site (IRES) is a cis‐acting element found in certain RNA viruses. In virus‐infected cells, the Senecavirus A (SVA) IRES can directly recruit the small ribosomal subunit to an internal initiation codon on the mRNA, enabling translation initiation independently of both the 5′ cap structure of the viral genome and the host cell eukaryotic initiation factor 4F (eIF4F). As a small RNA virus, SVA frequently accumulates genetic mutations during transmission. This study aimed to characterize the mutational and evolutionary patterns of the SVA genome during its transmission within host cells exhibiting enhanced innate immunity. SVA was serially passaged for 80 generations in preactivated 3D4/21 cells that had been established in an antiviral innate immune state. During serial passaging, two stable single‐nucleotide mutations were consistently identified in the IRES region of the viral genomic RNA from passages 60–80: a uridine (U) insertion at genomic position 109, and a guanine (G)‐to‐adenine (A) substitution at position 265. Furthermore, the rescued SVA mutants harboring these mutations exhibited significantly higher replication titers than the parental strain. Notably, our results indicated that the mutant virus was unable to evade host antiviral innate immunity, whereas it significantly enhanced the translational activity of the SVA IRES. Collectively, these findings provide a foundation for understanding how single nucleotide polymorphisms (SNPs) in the 5′ untranslated region (UTR) region influence viral IRES activity and the replication capacity of recombinant viruses.
Tao Li, Wen Dang, Weiwei Li et al.· Transboundary and Emerging D...· 0 citations
