QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.

SARS-CoV-2 Uses Nonstructural Protein 16 To Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'-O-methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'-O-MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo, using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive than wild-type SARS-CoV-2 to type I interferon (IFN-I) in vitro. Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'-O-methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, an MTase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment and attenuates viral replication. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a target for future antiviral therapies. IMPORTANCE Similar to other coronaviruses, disruption of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo, our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1 but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'-O-methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Covariation of viral recombination with single nucleotide variants during virus evolution revealed by CoVaMa.

Adaptation of viruses to their environments occurs through the acquisition of both novel single-nucleotide variants (SNV) and recombination events including insertions, deletions, and duplications. The co-occurrence of SNVs in individual viral genomes during their evolution has been well-described. However, unlike covariation of SNVs, studying the correlation between recombination events with each other or with SNVs has been hampered by their inherent genetic complexity and a lack of bioinformatic tools. Here, we expanded our previously reported CoVaMa pipeline (v0.1) to measure linkage disequilibrium between recombination events and SNVs within both short-read and long-read sequencing datasets. We demonstrate this approach using long-read nanopore sequencing data acquired from Flock House virus (FHV) serially passaged in vitro. We found SNVs that were either correlated or anti-correlated with large genomic deletions generated by nonhomologous recombination that give rise to Defective-RNAs. We also analyzed NGS data from longitudinal HIV samples derived from a patient undergoing antiretroviral therapy who proceeded to virological failure. We found correlations between insertions in the p6Gag and mutations in Gag cleavage sites. This report confirms previous findings and provides insights on novel associations between SNVs and specific recombination events within the viral genome and their role in viral evolution.

Next-generation sequencing: A new avenue to understand viral RNA-protein interactions.

The genomes of RNA viruses present an astonishing source of both sequence and structural diversity. From intracellular viral RNA-host interfaces to interactions between the RNA genome and structural proteins in virus particles themselves, almost the entire viral lifecycle is accompanied by a myriad of RNA-protein interactions that are required to fulfill their replicative potential. It is therefore important to characterize such rich and dynamic collections of viral RNA-protein interactions to understand virus evolution and their adaptation to their hosts and environment. Recent advances in next-generation sequencing technologies have allowed the characterization of viral RNA-protein interactions, including both transient and conserved interactions, where molecular and structural approaches have fallen short. In this review, we will provide a methodological overview of the high-throughput techniques used to study viral RNA-protein interactions, their biochemical mechanisms, and how they evolved from classical methods as well as one another. We will discuss how different techniques have fueled virus research to characterize how viral RNA and proteins interact, both locally and on a global scale. Finally, we will present examples on how these techniques influence the studies of clinically important pathogens such as HIV-1 and SARS-CoV-2.

ViReMa: a virus recombination mapper of next-generation sequencing data characterizes diverse recombinant viral nucleic acids.

BACKGROUND: Genetic recombination is a tremendous source of intrahost diversity in viruses and is critical for their ability to rapidly adapt to new environments or fitness challenges. While viruses are routinely characterized using high-throughput sequencing techniques, characterizing the genetic products of recombination in next-generation sequencing data remains a challenge. Viral recombination events can be highly diverse and variable in nature, including simple duplications and deletions, or more complex events such as copy/snap-back recombination, intervirus or intersegment recombination, and insertions of host nucleic acids. Due to the variable mechanisms driving virus recombination and the different selection pressures acting on the progeny, recombination junctions rarely adhere to simple canonical sites or sequences. Furthermore, numerous different events may be present simultaneously in a viral population, yielding a complex mutational landscape. FINDINGS: We have previously developed an algorithm called ViReMa (Virus Recombination Mapper) that bootstraps the bowtie short-read aligner to capture and annotate a wide range of recombinant species found within virus populations. Here, we have updated ViReMa to provide an "error density" function designed to accurately detect recombination events in the longer reads now routinely generated by the Illumina platforms and provide output reports for multiple types of recombinant species using standardized formats. We demonstrate the utility and flexibility of ViReMa in different settings to report deletion events in simulated data from Flock House virus, copy-back RNA species in Sendai viruses, short duplication events in HIV, and virus-to-host recombination in an archaeal DNA virus.

Zika Virus Infection Alters Gene Expression and Poly-Adenylation Patterns in Placental Cells.

Flaviviruses are small RNA viruses that are mainly transmitted via arthropod vectors and are found in tropic and sub-tropical regions. Most infections are asymptomatic (90-95%), but symptoms can be as severe as hemorrhagic fever and encephalitis. One recently emerged flavivirus is Zika virus (ZIKV), which was originally isolated from rhesus monkeys in Uganda roughly 70 years ago but has recently spread east, reaching S. America in 2015-2016. This outbreak was associated with the development of Guillain-Barré syndrome in adults and microcephaly in infants born to expectant mothers infected early in pregnancy. ZIKV must traverse the placenta to impact the development of the fetus, but the mechanisms responsible are unknown. While flaviviruses are known to disrupt splicing patterns in host cells, little is known about how flaviviruses such as ZIKV impact the alternative polyadenylation (APA) of host transcripts. This is important as APA is well-established as a mechanism in the regulation of mRNA metabolism and translation. Thus, we sought to characterize transcriptomic changes including APA in human placental (JEG3) cells in response to ZIKV infection using Poly(A)-ClickSeq (PAC-Seq). We used our differential Poly(A)-cluster (DPAC) analysis pipeline to characterize changes in differential gene expression, alternative poly-adenylation (APA) and the use of alternative terminal exons. We identified 98 upregulated genes and 28 downregulated genes. Pathway enrichment analysis indicated that many RNA processing and immune pathways were upregulated in ZIKV-infected JEG3 cells. We also updated DPAC to provide additional metrics of APA including the percentage-distal usage index (PDUI), which revealed that APA was extensive and the 3' UTRs of 229 genes were lengthened while 269 were shortened. We further found that there were 214 upregulated and 59 downregulated poly(A)-clusters (PACs). We extracted the nucleotide sequences surrounding these PACs and found that the canonical signals for poly-adenylation (binding site for poly-A binding protein (PABP) upstream and a GU-rich region down-stream of the PAC) were only enriched in the downregulated PACs. These results indicate that ZIKV infection makes JEG3 cells more permissive to non-canonical poly-adenylation signals.

SARS-CoV-2 Uses Nonstructural Protein 16 to Evade Restriction by IFIT1 and IFIT3.

Understanding the molecular basis of innate immune evasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important consideration for designing the next wave of therapeutics. Here, we investigate the role of the nonstructural protein 16 (NSP16) of SARS-CoV-2 in infection and pathogenesis. NSP16, a ribonucleoside 2'- O methyltransferase (MTase), catalyzes the transfer of a methyl group to mRNA as part of the capping process. Based on observations with other CoVs, we hypothesized that NSP16 2'- O MTase function protects SARS-CoV-2 from cap-sensing host restriction. Therefore, we engineered SARS-CoV-2 with a mutation that disrupts a conserved residue in the active site of NSP16. We subsequently show that this mutant is attenuated both in vitro and in vivo , using a hamster model of SARS-CoV-2 infection. Mechanistically, we confirm that the NSP16 mutant is more sensitive to type I interferon (IFN-I) in vitro . Furthermore, silencing IFIT1 or IFIT3, IFN-stimulated genes that sense a lack of 2'- O methylation, partially restores fitness to the NSP16 mutant. Finally, we demonstrate that sinefungin, a methyltransferase inhibitor that binds the catalytic site of NSP16, sensitizes wild-type SARS-CoV-2 to IFN-I treatment. Overall, our findings highlight the importance of SARS-CoV-2 NSP16 in evading host innate immunity and suggest a possible target for future antiviral therapies. Importance: Similar to other coronaviruses, disruption of SARS-CoV-2 NSP16 function attenuates viral replication in a type I interferon-dependent manner. In vivo , our results show reduced disease and viral replication at late times in the hamster lung, but an earlier titer deficit for the NSP16 mutant (dNSP16) in the upper airway. In addition, our results confirm a role for IFIT1, but also demonstrate the necessity of IFIT3 in mediating dNSP16 attenuation. Finally, we show that targeting NSP16 activity with a 2'- O methyltransferase inhibitor in combination with type I interferon offers a novel avenue for antiviral development.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called 'Tiled-ClickSeq', which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5'UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

Understanding Flavivirus Capsid Protein Functions: The Tip of the Iceberg.

Flaviviruses are enveloped positive-sense single-stranded RNA arboviruses, infectious to humans and many other animals and are transmitted primarily via tick or mosquito vectors. Capsid is the primary structural protein to interact with viral genome within virus particles and is therefore necessary for efficient packaging. However, in cells, capsid interacts with many proteins and nucleic acids and we are only beginning to understand the broad range of functions of flaviviral capsids. It is known that capsid dimers interact with the membrane of lipid droplets, aiding in both viral packaging and storage of capsid prior to packaging. However, capsid dimers can bind a range of nucleic acid templates in vitro, and likely interact with a range of targets during the flavivirus lifecycle. Capsid may interact with host RNAs, resulting in altered RNA splicing and RNA transcription. Capsid may also bind short interfering-RNAs and has been proposed to sequester these species to protect flaviviruses from the invertebrate siRNA pathways. Capsid can also be found in the nucleolus, where it wreaks havoc on ribosome biogenesis. Here we review flavivirus capsid structure, nucleic acid interactions and how these give rise to multiple functions. We also discuss how these features might be exploited either in the design of effective antivirals or novel vaccine strategies.