Unveiling the omicron B.1.1. 529: The variant of concern that is rattling the globe

Most viruses–including SARS-CoV-2, seem to have evolved over time. The lack of stringent proofreading mechanisms makes viral DNA/RNA replication error-prone. When a virus replicates, it sometimes changes a little bit, which is called mutations. Any virus with one or more new mutations can be referred to as a “variant” of the original virus. The last 2 years have witnessed the emergence of a large number of variants. Since the pandemic’s beginning, the SARS-CoV-2 coronavirus has mutated extensively, resulting in the emergence of different variants of the virus. One of these is the delta variant (arising from Pango lineage B.1.617.2) that took the word in a storm this year (February-July). The current a variant of concern is the B.1.1.529 (Omicron) variant reported first from South Africa on November 24, 2021. In recent weeks, infections have been widely reported, along with the increased detection of the B.1.1.529 variant. We reviewed the emergence of the new variant (B1.1.529) and its possible outcomes.

Relaxed Substrate Specificity in Qβ Replicase through Long-Term In Vitro Evolution

A change from RNA- to DNA-based genetic systems is hypothesized as a major transition in the evolution of early life forms. One of the possible requirements for this transition is a change in the substrate specificity of the replication enzyme. It is largely unknown how such changes would have occurred during early evolutionary history. In this study, we present evidence that an RNA replication enzyme that has evolved in the absence of deoxyribonucleotide triphosphates (dNTPs) relaxes its substrate specificity and incorporates labeled dNTPs. This result implies that ancient replication enzymes, which probably evolved in the absence of dNTPs, could have incorporated dNTPs to synthesize DNA soon after dNTPs became available. The transition from RNA to DNA, therefore, might have been easier than previously thought.

Multiscale Characterization Reveals Oligomerization Dependent Phase Separation of Primer-independent RNA Polymerase nsp8 From SARS-CoV-2

RNA replication and transcription machinery is an important drug target for fighting against coronavirus. Non-structure protein nsp8 was proposed harboring primase activity. However, the RNA primer synthesis mechanism of nsp8 is still largely unknown. Here, we purified dimer and tetramer forms of SARS-CoV-2 nsp8. Combined with DLS, SANS and thermo-stability analysis, we found that both dimer and tetramer become loosened and destabilized with decreasing salt concentration, and the dimer form is more stable than the tetramer form. Further investigation showed that nsp8 dimer and tetramer can undergo phase separation but exhibit different phase separation behaviors. nsp8 dimer can form liquid-like droplets in the buffer with a low concentration of NaCl; phase separation of nsp8 tetramer depends on the assistance of RNA. Our findings on different phase separation behaviors of nsp8 dimer and tetramer could provide novel insight into the primer synthesis mechanism in coronavirus and facilitate developing novel therapeutic agents against SARS-CoV-2.

Loquacious Modulates Flaviviral RNA Replication in Mosquito Cells

Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins SEC61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both SEC61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3’ end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.Author SummaryThere is a wealth of information that dictates virus-host interactions in flavivirus-infected mammalian cells, yet there is only sparse information on the mechanisms that modulate viral gene expression in the mosquito host. Using a novel RNA-protein detection assay, the interactions of SEC61A1 and Loqs with the dengue viral genome were found to have proviral functions in infected mosquito cells. In particular, Loqs forms complexes with the positive-strand of the viral RNA and the very 3’ end of the negative-strand viral RNA. Further analyses showed that Loqs modulates viral RNA replication of dengue virus and gene amplification of several other flaviviral genomes. These findings argue that Loqs is an essential proviral host factor in mosquitos.

Rolling Circle RNA Synthesis Catalysed by RNA

RNA-catalysed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5′ and 3′ ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

FUSE binding protein FUBP3 is a potent regulator in Japanese encephalitis virus infection

Background The JEV genome is a positive-sense RNA with a highly structured capped 5′UTR, 3′UTR and a large open reading frame. 3′UTR is the untranslated region of flavivirus and has various important functions during viral replication, such as translation, replication and encapsidation. During viral replication, the 3′UTR interacts with viral proteins and host proteins and is required for viral RNA replication and translocation. Methods The expression level of FUBP3 was knocked down by siRNA and Flag-tagged FUBP3 overexpression plasmid was constructed for overexpression. BHK-21 cells were cultured and infected with JEV to investigate the functional role of FUBP3 in the viral infection cycle. Subcellular localization of FUBP3 and viral replication complexes was observed by dual immunofluorescence staining. Results Four host proteins were specifically associated with the 3′UTR of JEV, and FUBP3 was selected to further investigate its potential functional role in the JEV infection cycle. Knockdown of FUBP3 protein resulted in a significant decrease in JEV viral titer, whereas ectopic overexpression of FUBP3 resulted in increased JE viral infectivity. In cells stably knocked down for FUBP3 and then infected with JEV, we found almost no detectable viral NS5 protein. In contrast, when cells stably knocking-down of FUBP3 overexpressed FUBP3, we found a significant increase in viral RNA production over time compared to controls. We also demonstrated that FUBP3 re-localized in the cytoplasm after infection with JEV and co-localized with viral proteins. Exogenous overexpression of FUBP3 was also shown to be located in the JE replication complex and to assist viral replication after JEV infection. Conclusions The overall results suggest that FUBP3 regulates RNA replication of JEV and promotes subsequent viral translation and viral particle production.

A conserved arginine in NS5 binds genomic 3’stem-loop RNA for primer-independent initiation of flavivirus RNA-replication

Replication of the RNA genome of flaviviruses without a primer involves RNA-protein interactions that have been shown to include the recognition of the stem-loop A (SLA) in the 5’ untranslated region (UTR) by the non-structural protein 5 (NS5). We show that DENV2 NS5 arginine 888, located within the C-terminal 18 residues, is completely conserved in all flaviviruses and interacts specifically with the top-loop of 3’SL in the 3’UTR which contains the pentanucleotide 5’-CACAG-3’ previously shown to be critical for flavivirus RNA replication. We present virological and biochemical data showing the importance of this Arg 888 in virus viability and de novo initiation of RNA polymerase activity in vitro. Based on our binding studies, we hypothesize that ternary complex formation of NS5 with 3’SL, followed by dimerization, leads to the formation of the de novo initiation complex that could be regulated by the reversible zipping and unzipping of cis-acting RNA elements.

The structure of a native orthobunyavirus ribonucleoprotein reveals a key role for viral RNA in maintaining its helical architecture

The Bunyavirales order of RNA viruses comprises emerging pathogens for which approved preventative or therapeutic measures for human use are not available. The genome of all Bunyavirales consists of negative-sense RNA segments wrapped by the virus-encoded nucleocapsid protein (NP) to form ribonucleoproteins (RNPs). RNPs represent the active template for RNA synthesis and the form in which the genome is packaged into virions, functions that require inherent flexibility. We present a pseudo-atomic model of a native RNP purified from Bunyamwera virus (BUNV), the prototypical Bunyavirales member, based on a cryo-electron microscopy (cryo-EM) average at 13 A resolution with subsequent fitting of the BUNV NP crystal structure by molecular dynamics. We show the BUNV RNP possesses relaxed helical architecture, with successive helical turns separated by ~18 A. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms, with no helix-stabilizing interactions along the longitudinal axis. Instead, EM analysis of RNase-treated RNPs suggests their chain integrity is dependent on the encapsidated genomic RNA, thus providing the molecular basis for RNP flexibility. Overall, this work will assist in designing anti-viral compounds targeting the RNP and inform studies on bunyaviral RNP assembly, packaging and RNA replication.