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PubMed2025

Enhanced RNA replication and pathogenesis in recent SARS-CoV-2 variants harboring the L260F mutation in NSP6.

The COVID-19 pandemic has been driven by SARS-CoV-2 variants with enhanced transmission and immune escape. Apart from extensive evolution in the Spike protein, non-Spike mutations are accumulating across the entire viral genome and their functional impact is not well understood. To address the contribution of these mutations, we reconstructed genomes of recent Omicron variants with disabled Spike expression (replicons) to systematically compare their RNA replication capabilities independently from Spike. We also used a single reference replicon and complemented it with various Omicron variant Spike proteins to quantify viral entry capabilities in single-round infection assays. Viral entry and RNA replication were negatively correlated, suggesting that as variants evolve reduced entry functions under growing immune pressure on Spike, RNA replication increases as a compensatory mechanism. We identified multiple mutations across the viral genome that enhanced viral RNA replication. NSP6 emerged as a hotspot with a distinct L260F mutation independently arising in the BQ.1.1 and XBB.1.16 variants. Using mutant and revertant NSP6 viral clones, the L260F mutation was validated to enhance viral replication in cells and increase pathogenesis in mice. Notably, this mutation reduced host lipid droplet content by NSP6. Collectively, a systematic analysis of RNA replication of recent Omicron variants defined NSP6's key role in viral RNA replication that provides insight into evolutionary trajectories of recent variants with possible therapeutic implications.

SARS-CoV-2↗
PubMed1991

Deletion analysis of brome mosaic virus 2a protein: effects on RNA replication and systemic spread.

Brome mosaic virus (BMV) genomic RNA2 encodes the 94-kDa 2a protein, which is one of two BMV nonstructural proteins required for RNA replication and subgenomic mRNA transcription. 2a contains a central polymeraselike region, which has extensive sequence similarity with the Sindbis virus nsP4 and tobacco mosaic virus (TMV) 183-kDa replication proteins, and also contains N- and C-terminal flanking segments without counterparts in the Sindbis virus and TMV nonstructural proteins. To further investigate the roles of the central and flanking segments in 2a, we have constructed a series of deletion and frameshift mutants in a biologically active BMV RNA2 cDNA clone and tested their ability to support viral RNA replication in barley protoplasts and systemic infection in whole barley plants. The entire 125-amino-acid C-terminal segment following the polymeraselike region was dispensable for RNA replication and transcription. Within the 200-amino-acid N-terminal flanking segment, deletion of the first 50 residues dramatically reduced genomic and subgenomic RNA accumulation, and deletion of 100 or more residues abolished detectable RNA synthesis. All mutations removing residues from the central polymeraselike domain also blocked RNA replication in trans. Sequences required in cis for RNA2 replication or stability were found to occur within the first 300 nucleotides of the 2a coding region. In whole barley plants, systemic infection was inhibited even by 2a deletions that supported strong RNA replication in protoplasts. Some replication-competent 2a variants failed to spread to uninoculated leaves, while other showed 10- to 500-fold-reduced virus yield in both inoculated and uninoculated leaves. These reductions were not due to any defects in RNA2 encapsidation.

Capsid↗
PubMed1992

Brome mosaic virus RNA replication proteins 1a and 2a from a complex in vitro.

Brome mosaic virus (BMV) is a positive-strand RNA virus that encodes two RNA replication proteins, the helicaselike 1a and the polymeraselike 2a. 1a and 2a share extensive sequence similarities with proteins encoded by many other members of the alphaviruslike superfamily. While further purifying enzymatically active RNA-dependent RNA polymerase from plants infected by BMV, we observed that 1a, 2a, and the polymerase activity all cofractionated through multiple independent purification steps. Moreover, using immunoprecipitation, we found that BMV 1a and 2a proteins synthesized in rabbit reticulocyte lysates or insect cells can form a specific complex in vitro. Complex formation was more efficient when 1a and 2a were cotranslated than when they were mixed after independent synthesis. In an antibody-independent assay, in vitro-translated 1a protein was also found to bind to 2a protein fixed on a nylon membrane. A three-amino-acid insertion in 1a that blocks BMV RNA replication in vivo also blocked in vitro interaction with 2a, while another two-amino-acid insertion that renders the 1a protein temperature sensitive for RNA replication interacted in vitro with 2a at 24 degrees C but not at 32 degrees C. These results and previous genetic data suggest that the 1a-2a interaction observed in vitro is required for BMV RNA replication and may have direct implications for other members of the alphaviruslike superfamily.

Amino Acid Sequence↗
PubMed1989

Vesicular stomatitis virus RNA replication: a role for the NS protein.

Synthesis of the vesicular stomatitis virus nucleocapsid (N) protein is required for viral RNA replication. The observation that the N protein forms a rapidly sedimenting species in the absence of other viral proteins and the description of complexes of N protein with NS protein led to the proposal that NS protein binds to N protein to prevent it from self-associating. We tested this model by analysing the physical properties of N protein synthesized alone in an in vitro replication system as compared to N protein synthesized in the presence of the NS protein. These findings were correlated with the ability of the N protein, synthesized under both conditions, to support replication. N protein synthesized at low concentrations in the absence of other viral proteins sedimented at 4S on glycerol gradients and was capable of supporting RNA replication. In contrast, synthesis of increasing concentrations of N protein resulted in formation of a rapidly sedimenting species of N protein which had the physical properties of a protein-protein aggregate and which failed to support RNA replication. Co-synthesis of the NS protein with N protein both prevented the concentration-dependent aggregation of N and restored the ability of high concentrations of N protein to support RNA replication.

Capsid↗
PubMed1983

Kinetics of RNA replication.

The reaction kinetics of single-stranded RNA replication were investigated by means of analytical and computer simulation methods. A model reaction mechanism is proposed that is in accord with the extensive experimental data available for the replication of various templates by the enzyme Q beta replicase. Despite the complexity of this mechanism, conventional concepts of steady-state and dynamic enzyme catalysis and plausible values of the rate and stability constants for the elementary reactions suffice to provide detailed understanding of RNA replication kinetics. The main features can be described with simple formulas that are analogous to traditional descriptions of enzyme kinetics.

Genes, Viral↗
PubMed1973

Two classes of membrane binding of replicative RNA of bacteriophage MS2.

Escherichia coli membranes were isolated in the presence of 6 mM Mg(++). They were washed with buffer containing no Mg(++) to yield a fraction containing material bound only in the presence of divalent cations, "membrane eluate," and that bound in the absence of divalent cations, "membrane." When E. coli infected with bacteriophage MS2 are labeled with [(14)C]uracil, all MS2 replicative RNA, i.e., the RNA species containing MS2 complementary RNA, is in the membrane eluate and membrane. The amount of [(14)C]uracil in replicative RNA found in the membrane eluate increases with time of labeling, whereas that in the replicative RNA in the membrane reaches a plateau in 1-2 min. This finding is consistent with a precursor-product relationship. Most of the label entering single-stranded viral RNA comes from the replicative RNA in the membrane eluate. This result suggests that polymerase components or factors required for complementary-strand synthesis are bound to membrane even in the absence of divalent cations and that the polymerase is no longer bound to these factors when it is making the bulk of the progeny single-stranded RNA.

Binding Sites↗
PubMed1987

A new 'sense' RNA approach to block viral RNA replication in vitro.

The use of "antisense" RNA is being widely considered to block specific steps in viral infection. We propose here a new "sense" RNA approach to block viral RNA replication in vitro and possibly in vivo. In the turnip yellow mosaic virus (TYMV) system, the recognition site of the viral replicase (RNA-dependent RNA polymerase) is assumed to be located within the 3' end of the RNA genome. Small "sense" RNAs have been obtained by in vitro transcription of the corresponding cloned cDNAs. Replication of TYMV RNA in vitro is shown here to be blocked only by those RNAs that contain the 3' terminal region of the genome.

Antiviral Agents↗
PubMed1984

Synthesis of the templates for influenza virion RNA replication in vitro.

To elucidate the mechanism(s) of influenza viral RNA replication, we have developed an in vitro system in which the templates for viral RNA replication as well as the viral messenger RNAs (mRNAs) are synthesized. Because the synthesis of both the viral mRNAs and the template RNAs occurs in the nucleus of infected cells, we determined whether infected cell nuclei are active in the synthesis of these two types of transcripts in vitro. Nuclei isolated as early as 1-2 hr after infection catalyze the in vitro synthesis of both the viral mRNAs and template RNAs. The time course of appearance of these activities indicates that they most likely represent the transcriptional complexes functioning in vivo. Template RNA synthesis catalyzed by the nuclei in vitro is independent of concomitant protein synthesis; rather, it utilizes preformed proteins present in the nuclear preparations. This protein pool can be depleted by treating the infected cells with a protein synthesis inhibitor prior to the isolation of the nuclei, thereby rendering the nuclei inactive in template RNA synthesis in vitro. This activity can be restored by the addition of infected cell cytoplasmic extracts or of the high-speed supernatant fraction from these extracts. These results indicate that the cytoplasmic fraction from infected cells enables the viral transcription complex to continue transcription past the site at which termination occurs during viral mRNA synthesis and also suggest that this fraction enables the transcription complex to initiate transcription without the capped primer used in viral mRNA synthesis.

Animals↗
PubMed1984

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.

Animals↗
PubMed1983

Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides.

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have also revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 and P3 regions of the polyprotein. Sequence analysis of stepwise Edman degradation show that these proteins are 3b/9 (Mr 77,000) and X/9 (Mr 50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.

Amino Acid Sequence↗
PubMed1995

RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA.

Previously, a cDNA was constructed so that transcription by T7 RNA polymerase yielded a approximately 1-kb negative-sense analog of genomic RNA of human respiratory syncytial virus (RSV) containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative RSV transcription motifs and flanked by the RSV genomic termini. When transfected into RSV-infected cells, this minigenome was "rescued," as evidenced by high levels of CAT expression and the production of transmissible particles which propagated and expressed high levels of CAT expression during serial passage (P.L. Collins, M. A. Mink, and D. S. Stec, Proc. Natl. Acad. Sci. USA, 88:9663-9667, 1991). Here, this cDNA, together with a second one designed to yield an exact-copy positive-sense RSV-CAT RNA antigenome, were each modified to contain a self-cleaving hammerhead ribozyme for the generation of a nearly exact 3' end. Each cDNA was transfected into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase, together with plasmids encoding the RSV N, P, and L proteins, each under the control of a T7 promoter. When the plasmid-supplied template was the mini-antigenome, the minigenome was produced. When the plasmid-supplied template was the minigenome, the products were mini-antigenome, subgenomic polyadenylated mRNA and progeny minigenome. Identification of progeny minigenome made from the plasmid-supplied minigenome template indicates that the full RSV RNA replication cycle occurred. RNA synthesis required all three RSV proteins, N, P, and L, and was ablated completely by the substitution of Asn for Asp at position 989 in the L protein. Thus, the N, P, and L proteins were sufficient for the synthesis of correct minigenome and antigenome, but this was not the case for subgenomic mRNA, indicating that the requirements for RNA replication and transcription are not identical. Complementation with N, P, and L alone yielded an mRNA pattern containing a large fraction of molecules of incomplete, heterogeneous size. In contrast, complementation with RSV (supplying all of the RSV gene products) yielded a single discrete mRNA band. Superinfection with RSV of cells staging N/P/L-based RNA synthesis yielded the single discrete mRNA species. Some additional factor supplied by RSV superinfection appeared to be involved in transcription, the most obvious possibility being one or more additional RSV gene products.

Bacteriophage T7↗
PubMed1975

The host factor required for RNA phage Qbeta RNA replication in vitro. Intracellular location, quantitation, and purification by polyadenylate-cellulose chromatography.

The Qbeta host factor, a heat-stable protein necessary in concert with Qbeta replicase for phage Qbeta RNA replication in vitro, has been localized in Escherichia coli and found to be associated primarily with ribosomes. This location has been established both by complement fixation assays with highly specific antiserum directed against the host factor, and by in vitro stimulation of Qbeta RNA replication by the Qbeta replicase. The complement fixation assay has provided the estimate that there are approximately 2500 copies of the host factor polypeptide per cell. The host factor is released from the ribosomes by a 1 M NH4Cl wash and concentrated by ammonium sulfate precipitation. It can be purified to apparent homogeneity in one further step by chromatography on poly(A)-cellulose. Ribosomal protein S1 subunit I of Qbeta replicase) also binds to the poly(A)-cellulose column and elutes before the host factor. In agreement with previous reports, we find that the host factor has a monomer molecular weight of 12,000 as judged by sodium dodecyl sulfate-polyacrylamide gels, and a native molecular weight of 72,000 as judged by the stoichiometric interaction of the host factor with Qbeta RNA, by sedimentation in sucrose velocity gradients, and by sodium dodecyl sulfate gel mobility when incompletely disaggregated. The Qbeta host factor is a potent inhibitor of an in vitro poly(A)-directed polylysine protein-synthesizing system, but has less effect on the in vitro translation of poly(U), R17 RNA, late T7 mRNA, or endogenous E. coli mRNA. The amino acid composition and NH2- terminal sequence rule out the host factor as one of the known 30 S or 50 S E. coli ribosomal proteins. The finding that the Qbeta host factor is associated with ribosomes in vivo completes the demonstration that all of the host-supplied proteins required for phage Qbeta RNA replication in vitro are either associated with ribosomes or are involved in the protein-synthetic machinery of the cell.

Animals↗
PubMed1983

Effect of an Escherichia coli traD (ts) mutation on MS2 RNA replication.

We have continued our studies on the effect of high temperature (42 degrees C) on the development of RNA bacteriophage MS2 in the temperature-sensitive conjugational transfer-deficient mutant Escherichia coli JCFL39 carrying a traD (ts) mutation. At 42 degrees C, mutant cells permit the penetration and translation of phage MS2 RNA but do not permit MS2 RNA replication. We suggest a role for the traD (ts) mutation in MS2 RNA replication.

Coliphages↗
PubMed1987

Action of 3-methylquercetin on poliovirus RNA replication.

3-Methylquercetin is a natural flavone that powerfully blocks poliovirus replication. This compound inhibits selectively poliovirus RNA synthesis both in infected cells and in cell-free systems. Poliovirus double-stranded RNA (replicative forms) is still made in the presence of this inhibitor, whereas the synthesis of single-stranded RNA and the formation of replicative intermediates are drastically blocked.

Antiviral Agents↗
PubMed1994

Nodavirus RNA replication: mechanism and harnessing to vaccinia virus recombinants.

In order to harness RNA replication for the amplification of mRNAs expressed from recombinant vectors and vaccines, we constructed a VV recombinant that expressed the RNA replicase encoded in the larger genomic segment of the nodavirus FHV. When both termini of the VV-derived transcript were correct, the encoded enzyme replicated its own mRNA, and replication dominated the RNA synthetic capacity of the cell. The smaller genomic segment of FHV could also be replicated by the enzyme when supplied in trans, either by coinfection with another VV recombinant or by transfection of an appropriate plasmid. However, two requirements had to be fulfilled for replication of the smaller FHV RNA segment. The first was the prior replication of the larger genomic segment, which was interpreted as a mechanism to achieve sufficient replicase synthesis before the onset of coat protein synthesis. The second was the presence in the smaller genomic RNA of an internal region between about nucleotides 525-620. Work is in progress to elucidate the reasons for these requirements for RNA 2 replication.

Animals↗
PubMed1992

Mechanism of coronavirus transcription: duration of primary transcription initiation activity and effects of subgenomic RNA transcription on RNA replication.

Previously, we established a system whereby an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells. By using this system, the duration of a primary transcription initiation activity which transcribes subgenomic-size RNAs from the genomic-size RNA template in MHV-infected cells was examined. Efficient DI genomic and subgenomic RNA synthesis was observed when the DI RNA was transfected at 1, 3, 3.5, 5, and 6 h postinfection, indicating that all activities which are necessary for MHV RNA synthesis are present continuously during the first 6 h of infection. The effect of subgenomic DI RNA synthesis on DI genomic RNA replication was then examined. Replication efficiency of the DI genomic RNA which synthesized the subgenomic RNA was approximately 70% lower than that of DI genomic RNA which did not synthesize the subgenomic DI RNA in MHV-infected cells. Cotransfection of two different-size DI RNAs demonstrated that replication of the larger DI RNA was strongly inhibited by replication of the smaller genomic DI RNA. Cotransfection of two DI RNA species of the same length into MHV-infected cells demonstrated that reduced replication of the genomic DI RNA which synthesizes the subgenomic RNA did not affect the replication of cotransfected DI RNA, demonstrating that the reduction in DI genomic RNA replication works only in cis, not in trans. Therefore, the previously proposed hypothesis that coronavirus, subgenomic RNA synthesis may inhibit the replication of genomic RNA by competing for a limited amount of virus-derived factors seems unlikely. Possible mechanisms of coronavirus transcription are discussed.

Animals↗
PubMed1989

Analysis of sequences involved in cowpea mosaic virus RNA replication using site-specific mutants.

Using a full-length cDNA clone of cowpea mosaic virus (CPMV) B-RNA from which infectious transcripts can be generated, we examined the influence of a sequence of 11 nucleotides, UUUUAUUAAAA, comprising the nucleotides 5883 to 5893 in the 3' noncoding region of B-RNA, on viral RNA replication. This sequence is not only present in B-RNA but also in M-RNA and represents the 7 nucleotides preceding the poly(A) tail and the first four A residues of the poly(A) tail. Replication of B-RNA transcripts derived from a series of mutants in this region was tested in cowpea plants and protoplasts. Only mutant transcripts with minor modifications appeared able to replicate, which indicates that the region has a function in viral RNA replication. In addition, the results suggest the existence of a hairpin loop in this region. Those transcripts with deletions which disturb the putative hairpin structure have decreased specific infectivities. Mutant transcripts reversed stepwise to the wild-type sequence during replication in plants. This observation strengthens the idea that the sequence of 11 nucleotides has a function in viral RNA replication.

Base Sequence↗