Complementary Minus Strand Research Articles

SuperSelective primer pairs for sensitive detection of rare somatic mutations

SuperSelective primers, by virtue of their unique design, enable the selective exponential amplification of rare DNA fragments containing somatic mutations in the presence of abundant closely related wild-type DNA fragments. However, when a SuperSelective primer is used in conjunction with a conventional reverse primer, linear amplification of the abundant wild-type fragments occurs, and this may lead to a late arising signal that can be confused with the late arising signal from the rare mutant fragments. We have discovered that the use of a pair of SuperSelective primers, one specific for the target mutation in a plus strand, and the other specific for the same mutation in the complementary minus strand, but both possessing 3′-terminal nucleotides that are complementary to the mutation, significantly suppresses the linear amplification of the related wild-type sequence, and prevents the generation of false mutant sequences due to mis-incorporation by the DNA polymerase. As a consequence, the absence of mutant fragments in a sample does not give rise to a false-positive signal, and the presence of mutant fragments in a sample is clearly distinguishable as a true-positive signal. The use of SuperSelective primer pairs should enhance the sensitivity of multiplex PCR assays that identify and quantitate somatic mutations in liquid biopsies obtained from patients with cancer, thereby enabling the choice of a targeted therapy, the determination of its effectiveness over time, and the substitution of a more appropriate therapy as new mutations arise.

Duplex formation between the template and the nascent strand in the transcription-regulating sequences is associated with the site of template switching in SARS – CoV-2

ABSTRACT Recently published transcriptomic data of the SARS-CoV-2 coronavirus show that there is a large variation in the frequency and steady state levels of subgenomic mRNA sequences. This variation is derived from discontinuous subgenomic RNA synthesis, where the polymerase switches template from a 3ʹ proximal genome body sequence to a 5ʹ untranslated leader sequence. This leads to a fusion between the common 5ʹ leader sequence and a 3ʹ proximal body sequence in the RNA product. This process revolves around a common core sequence (CS) that is present at both the template sites that make up the fusion junction. Base-pairing between the leader CS and the nascent complementary minus strand body CS, and flanking regions (together called the transcription regulating sequence, TRS) is vital for this template switching event. However, various factors can influence the site of template switching within the same TRS duplex. Here, we model the duplexes formed between the leader and complementary body TRS regions, hypothesizing the role of the stability of the TRS duplex in determining the major sites of template switching for the most abundant mRNAs. We indicate that the stability of secondary structures and the speed of transcription play key roles in determining the probability of template switching in the production of subgenomic RNAs. We speculate on the effect of reported variant nucleotide substitutions on our models.

Identification of Potential Key Agents for Targeting RNA-Dependent RNA Polymerase of SARS-CoV-2 by Integrated Analysis and Virtual Drug Screening.

BackgroundRNA-dependent RNA polymerase (RdRp) is the key enzyme responsible for the SARS-CoV-2 replication process and catalyzes the synthesis of complementary minus strand RNA and genomic plus strand RNA, often recognized as good targets for antiviral drugs.Materials and MethodsA systematic screening of existing antiviral compounds, family analysis, conserved domain analysis, three-dimensional structure modeling, drug virtual screening, and bioassays were performed to identify agents that potentially targeted RNA-dependent RNA polymerase of SARS-CoV-2.ResultsFour thousand nine hundred and forty seven antiviral lead compounds were selected and evaluated by systematic screening. Of these, 359 agents were screened by family analysis and conserved domain analysis. They were further analyzed by three-dimensional structure modeling, virtual drug screening, and bioassays. The results identified 102 agents with potential for repurposing to target the RNA-dependent RNA polymerase of SARS-CoV-2.ConclusionThis study identified 102 key agents with potential anti-SARS-CoV-2 RNA-dependent RNA polymerase function and prospects of rapid clinical application for the treatment of COVID-19.

Properties and use of novel replication-competent vectors based on Semliki Forest virus

BackgroundSemliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important.ResultsWe analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture.ConclusionThe properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.

Northern Analysis of Viral Plus‐ and Minus‐Strand RNAs

Replication is a fundamental activity of viruses. Replication of positive-sense RNA viruses involves the synthesis of complementary minus-strand intermediates from the parental RNA template followed by synthesis of nascent plus strands. Negative-sense RNA genome and double-stranded RNA are copied into positive-sense mRNA before translation. To detect and estimate the abundance of plus- and minus-strand viral transcripts in the infected samples, northern analysis is the most commonly used method.

The Murine Charged Multivesicular Body Protein 2A, CHMP2A Interacts with the 5’ and 3’ Terminal Regions of Dengue Virus Complementary Minus-strand RNA

Dengue (DEN) viruses, of which there are four distinct serotypes (DEN-1, -2, -3 and -4), belong to the Flaviviridae family. Their 'plus' sense RNA genomes contain Non-Translated Regions (NTRs) at their 5' and 3' ends. Replication of viral genomic RNA, which takes place in close association with host cell membranes, involves the initial synthesis of the complementary minus sense RNA intermediate. The NTRs, which have the potential to form stable stem-loop structures, are implicated to play a key role in the viral life cycle through binding to viral and/or host proteins. We have screened a mouse macrophage cDNA expression library with a mixture of radioactive plus and minus sense DEN-2 virus NTR transcripts and identified a ~25 kDa protein. A BLAST analysis of the cDNA sequence encoding this protein showed it to be identical to human CHarged Multivesicular body Protein 2A, CHMP2A (also known as CHromatin Modifying Protein 2A), implicated in sorting proteins into endosome-derived vesicles. Recombinant murine CHMP2A was expressed in E. coli as a 6x His tagged protein, purified to homogeneity and shown to interact preferentially with the minus sense 5' and 3' NTRs of all four DEN virus serotypes. Using DEN-2 virus 5' (-) and 3' (-) NTRs we could demonstrate the specificity of this binding activity in vitro by ultraviolet cross-linking and electrophoretic mobility shift assays, carried out in the absence and presence of specific and non- specific competitors. Finally, this unique RNA/protein interaction was verified in vivo using a yeast- based interaction assay.

Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg.

The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3D(pol), and Mg(2+) (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.

Secondary structure of the 3' untranslated region of flaviviruses: similarities and differences

Genetic algorithm-based RNA secondary structure prediction was used in combination with comparative sequence analysis to construct models of folding for the distal part of the 3'-untranslated region of flaviviruses belonging to four serological groups. Elements of RNA secondary structure that are preserved among all the flaviviruses studied were revealed, despite the high degree of sequence divergence between them. At the same time, structural elements were observed that distinguish members of different serological groups and, in particular, a region of remarkable structural divergence between the tick-borne and mosquito-borne flaviviruses was found. Application of the genetic algorithm also revealed that the 3'-terminus of flaviviral genomic RNA may take on alternative conformations, which are not observed in the 3'-terminus of complementary minus strand RNA. These alternative folding patterns may have roles in the regulation of transcription and translation initiation and in the switch between them.

Anin VitroSystem for the Investigation of Heterologous RNA Recombination

Bacteriophage Φ6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. Purified procapsids are capable of the specific packaging of viral plus strands and the synthesis of their complementary minus strands. The genomic segments of Φ6 are capable of heterologous recombination. We have prepared anin vitrosystem containing purified procapsids that is capable of packaging plus strands of the genomic segments and synthesizing minus strands on these templates. The system generates heterologous recombination products when stimulated by having one of the plus strands incapable of serving as a template for minus strand synthesis. Recombinants were produced upon transfection of spheroplasts with thein vitropackaged and replicated RNA. Sites of recombination were not found to be localized in particular regions of either the donor or the recipient strands.

Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.

Double-Stranded Strong-Stop DNA and the Second Template Switch in Human Immunodeficiency Virus (HIV) DNA Synthesis

Synthesis of unintegrated retroviral DNA via reverse transcription is thought to involve two separate template switches. Minus-strand strong-stop DNA, a major product of in vitro reactions using detergent-treated virions, is synthesized prior to the first template switch. Plus-strand strong-stop DNA, which is found base-paired to near full-length minus-strand DNA in infected cells, is believed to be synthesized before the second template switch. Using a synchronized, one-step HIV infection model, we report here the detection in acutely infected cells of a novel double-stranded strong-stop HIV DNA with a discrete length of ∼650 base pairs, commencing at or near the left hand end of the right-hand U3 region of the HIV long terminal repeat. The plus-strand of this double-stranded strong-stop DNA possesses the primer binding site sequence and appears to be synthesized prior to the completion of the synthesis of its complementary minus-strand. In contrast, the minus-strand of the double-stranded strong-stop DNA lacks the primer binding site sequence after RNaseH digestion. We propose that a transient free plus-strand strong-stop DNA is released from its template by displacement synthesis and subsequently used as template for the synthesis of its complementary minus-strand. The proposed transient free plus-strand strong-stop DNA may also mediate the second template switch.

Protein P4 of the bacteriophage phi 6 procapsid has a nucleoside triphosphate-binding site with associated nucleoside triphosphate phosphohydrolase activity.

Bacteriophage phi 6 contains three segments of double-stranded RNA. The procapsid consists of proteins P1, P2, P4, and P7, which are encoded by the viral L segment. cDNA copies of this segment have been cloned into plasmids that direct the production of these proteins, which assemble into polyhedral procapsids. These procapsids are capable of packaging plus-sense phi 6 RNA in the presence of nucleoside triphosphate and synthesizing the complementary minus strand to form double-stranded RNA. In this article, we report the presence of a nucleotide-binding site in protein P4. The viral procapsid and nucleocapsid exhibit a nucleoside triphosphate phosphohydrolase activity that converts nucleoside triphosphates into nucleoside diphosphates.

Effects of 2-chloroadenine substitution in DNA on restriction endonuclease cleavage reactions.

The purine analog, 2-chloro-2'-deoxyadenosine triphosphate (CldATP), was incorporated enzymatically in place of dATP into the minus strand of M13mp18 duplex DNA. Its effect on protein-DNA interactions was assessed by determining the amount of DNA cleavage by type II restriction endonucleases. Substitution of chloroadenine (CIAde) for adenine (Ade) in DNA appreciably decreased the amount and rate of DNA cleavage of the minus strand when the analog was situated within the appropriate endonuclease recognition site. CIAde residues flanking a restriction site had variable effects. SmaI cleaved both CIAde-containing and control substrates with equal efficiency. NarI, however, was stimulated 1.5-fold by the presence of CIAde outside its recognition site. The effects of analog incorporation on restriction enzyme cleavage of an opposing unsubstituted strand of duplex DNA was examined by enzymatically incorporating CIdATP into the complementary minus strand of a 36-base oligonucleotide. Endonucleolytic cleavage of both plus and minus strands was reduced on 36-mers containing CIAde residues located within only the minus strand. These data suggest that CIAde residues incorporated into a single DNA strand may have an appreciable effect on DNA-protein interactions that involve one or both strands of duplex DNA.

Electrophoretic separation of the plus and minus strands of rotavirus SA11 double-stranded RNAs

The genome of the rota viruses consists of eleven segments of completely double-stranded RNA (dsRNA). To provide a method for separating and identifying the complementary plus and minus strand RNAs within these segments, we have characterized their migration patterns under denaturing conditions on agaroseurea gels. Virion-derived 3H-labelled dsRNAs were resolved by electrophoresis on a polyacrylamide gel and the individual genome segments recovered by electroelution. Upon electrophoresis in a low pH 1.75% agarose gel containing 6 M urea, the dsRNAs were denatured with complementary plus and minus strands migrating at different rates. Our results showed that, like cytoplasmic polyhedrosis virus but unlike human reovirus (Smith et al., 1981), rotavirus plus strand RNA migrates faster than its complementary minus strand on agarose-urea gels.

Virus infection as a function of the host cell life cycle: replication of poliovirus RNA.

SUMMARY The rates of synthesis and final yields of poliovirus RNA varied considerably during the four main phases (G1, S, G2 and m) of the life cycle of synchronized HeLa cells. The rate of RNA synthesis late in virus growth (as measured by uridine incorporation 2 to 4 hr after infection) and the final yield of RNA rose sharply if growth was initiated towards the end of phase S; that is, RNA was synthesized most rapidly if cells were infected during the period of most rapid DNA synthesis. In contrast, the initial rate of RNA synthesis (incorporation 0 to 2 hr after infection) was greatest if growth was initiated at the end of phase G2, just before mitosis. This differential effect on growth kinetics suggests that the balance between the two stages of virus RNA synthesis (production of complementary minus strands and of progeny plus strands) is dependent on unknown cellular factors.

Mechanism of reovirus double-stranded ribonucleic acid synthesis in vivo and in vitro.

The complementary strands of reovirus double-stranded ribonucleic acid (ds RNA) are synthesized sequentially in vivo and in vitro. In both cases, preformed plus strands serve as templates for the synthesis of the complementary minus strands. The in vitro synthesis of dsRNA is catalyzed by a large particulate fraction from reovirus-infected cells. Treatment of this fraction with chymotrypsin or with detergents which solubilize cellular membranes does not alter its capacity to synthesize dsRNA. The enzyme or enzymes responsible for dsRNA synthesis remain sedimentable at 10,000 x g after these enzyme or detergent treatments, indicating their particulate nature. Pretreatment of this fraction with ribonuclease, however, abolishes its ability to catalyze dsRNA synthesis, emphasizing the single-stranded nature of the template and its location in a structure permeable to ribonuclease. In contrast, the newly formed dsRNA is resistant to ribonuclease digestion at low salt concentrations and hence is thought to reside within a ribonuclease-impermeable structure.

Resolution of two factors required in the Q-beta-RNA polymerase reaction.

THE Escherichia coli bacteriophage Qβ contains RNA as its genetic component. Qβ-RNA can be synthesized in vitro in the reaction catalysed by the Qβ-RNA polymerase isolated from Qβ infected E. coli1. The synthesis of infectious Qβ-RNA requires, in addition to the polymerase, the phage RNA to serve as template, ribonucleoside tri-phosphate substrates, Mg++, and a factor fraction obtained from both infected and uninfected E. coli2. Previous studies with the factor have suggested that it acts on Qβ-RNA at some early step in the reaction2. Although an association of the enzyme and Qβ-RNA occurs in the absence of this factor3, synthesis of the complementary minus strand is not detected. The requirement for this agent in the reaction seems to bear a quantitative relationship to Qβ-RNA but not to enzyme2. The factor fraction, furthermore, is not required for enzyme activity when synthetic polymers4, minus strands or other RNA molecules3 are used as template. These results suggest that the role of factor is to promote a step in the reaction occurring after association of the enzyme with Qβ-RNA, but before nucleotide polymerization.