Viral Minus strand

Barrijal S, Perros M, Gu Z, Avalosse BL, Belenguer P, Amalric F, Rommelaere J (1992) Nucleolin forms a specific complex with a fragment of the viral (minus) strand of minute virus of mice DNA. Nucleic Acids Res 20 5053-5060... 

RNA viruses that cause tumors (oncogenic RNA viruses) are called retroviruses because their life cycle involves a DNA intermediate. The ability of retroviruses to use such a route for replication hinges on a viral-encoded enzyme called reverse transcriptase, an enzyme with three discrete activities (1) It catalyzes the synthesis of DNA from the viral plus-strand (2) it catalyzes the synthesis of DNA plus-strand from the viral minus-strand DNA and (3) it catalyzes the degradation of the viral RNA from an RNA-DNA heteroduplex. 

Fig. 8.4 Hypercycle phenomena can be observed when a cell is infected by an RNA virus. The vims provides the host cell with information for an enzyme favouring only the reproduction of viral information, i.e., of an RNA strand. This RNA is converted by the host cell into a protein (a replicase) which forms a new RNA minus-strand. The latter is then replicated to give a plus-strand (Eigen et al., 1982)...

Figure 5. The mechanism of RNA replication by means of viral-specific RNA repli-cases. The sketch shows a simplified version of the mechanism of RNA replication by QP replicase. The mechanism within each of the two cycles consists of binding of the RNA (l+ and I" standing for plus- or minus-strand respectively) to the enzyme (E), elongation of the growing chain (see Figure 2) and, eventually, dissociation of the enzyme-RNA complexes. Rate constants for association, elongation and dissociation are indicated by kX, kf, kp, k), ki and kp, respectively. According to complementar-...

DNA-directed DNA polymerase activity This activity replicates the minus strand DNA to yield dsDNA after viral RNA genome is degraded. The polymerase domain of HIV-1 RT shares structural similarity with other polymerases, presumably catalyzing the polymerization with the same reaction mechanism (Steitz, 1993 Steitz, 1998). 

The analysis of polio RI for VPg did not permit us to decide whether YPg was also linked to polio minus strands, since the quantitation of YPg is too inaccurate and because at 3 5 hr after infection, 90% of the RI structures produce plus strands (32). Evidence that TPg was linked to the 5 "te2 minal poly(u) of minus strands of RI as well as viral double-stranded RNAs (RF) was obtained by an analysis of hybrid double-stranded composed of 52p-labeled minus strands and unlabeled plus strands (I3). (52p ) poly(u) was isolated from these hybrids and had a 5 -terminal pU linked to YPg (13) This result has recently been confirmed by Pettersson al. (30 It has also been extended by Wu al. (15)> who derivatized the YPg of polio RF with dinitrofluorobenzene and subsequently complexed it with anti-DNP IgG. Inspection of these molecules in the electron microscope showed that both ends of RF were protein-tagged. The frequency of tagging was high enough to conclude that both strands of polio RF are protein-linked. 

If idENA has the same nucleotide sequence as virion MA, why-are mRNA strands not encapsidated into virions A trivial explanation would be that viral MA, once it has combined with ribosomes, may subsequently not exist free of them. However, free viral MA (EHA not bound to ribosomes) that is 5 "terminated with pUp has been found in cytoplasmic extracts of infected cells (21). Thus there exists the intriguing possibility that, in addition to the role in replication, VPg linked to the 5 end of plus-strand EHA is also a prerequisite for virion formation. For example, the newly synthesized Pg-MA may associate with one or several capsid protomers (42) prior to encapsidation. This protomer/VPg-EHA complex could then protect the YPg(Tyr)-0 pU linkage from cleavage by the host cell enzyme. Consequently, the concentration of protomers would determine how much of the newly synthesized VPg-RNA is "processed" to mENA, that is, early in infection most if not all progeny EHA may be cleaved at the 5 end. If VPg linked to EHA is a prerequisite for encapsidation, why do virions not contain minus strands A simple explanation could be provided by the observation that newly synthesized minus strands are rapidly incorporated into El and RP structures inside the membrane-bound replication complex minus strands are not found as free ENA in the cytoplasm (5) and are thus neither cleaved at the 5 end nor encapsidated. 

Replication of the viral RNA is a two-step transcriptional process, involving the synthesis of a complementary ("minus") strand as a first step, followed by the synthesis of new RNA chains of the original polarity, using this time the "minus" strand as template. 

The poly(A) stretch directs transcription of a poly(U) tract at the 5 end of the "minus" strand, which in turn serves as template to reproduce the poly(A) tail in the newly synthesized viral RNA (59> 87-89) Thus, unlike the poly(A) tract of eukaryotic mRNAs, the poly(A) stretch of picornavirus RRA is not post-transcriptionally added but is genetically coded. 

As synthesis of RNA proceeds in the 5 to 3 direction, transcription of the viral RNA must initiate at the 3 end of the template, that is, on the poly(A) tract. A poly(B) polymerase hence appears quite likely to be the enzyme responsible for initiating the synthesis of the minus strand. The need for an oligo(u) primer, however, makes that enzyme dependent on another one (be it of cellular or viral origin) which in t irn must provide the primer. Alternatively, the requirement of a pre-formed oligo(u) primer might simply reflect the inability of the m vitro system to initiate transcription in the absence of a UTP residue linked to the small viral protein VPg (chapter 9) ... 

Minus strand In viral genomes, a nucleic acid strand that is complementary to the RNA strand that serves as mRNA. Compare plus strand. 

Feix, G., Pollet, R. and Weissmann, C. (1968) Replication of viral RNA, XVI. Enzymatic synthesis of infectious viral RNA with noninfectious QjS minus strands as template. Proc. Nat. Acad. Sci. U.S.A. 59, 145-152. 

Plus Strand In viral genomes, a nucleic add strand that can serve as mRNA or (for DNA strand) that is homologous to one that can as distinct from the complementary (minus) strand. Most viruses with single-strand genomes package only the plus or minus strand in virions the other strand is made transiently during replication. Compare Minus Strand. 

Major distinct problems for experimental study in the near future include (1) the question of whether both types of virions are capable of or necessary for productive infection (2) a mechanism of DNA replication which will account for the fine structure of the DNA and the observation that approximately equal numbers of plus and minus strands are separately encapsidated (3) determination of the portion of the amino acid sequence of the structural proteins which can be or are coded for by the viral genome (4) the manner of association of the viral genome with latently infected cells and (5) further studies on the defectiveness of AAV. 

These results indicate that the poliovirus RF-RNA served as a template for the synthesis of RNA complementary to viral RNA (i.e. minus-strand RNA). Separated poliovirus minus- and plus-strand RNAs anneal with an efficiency of 60 % under our experimental conditions (RNA concentrations of 5 (xg/ml, assay of RNase resistance with Tj RNase and RNase I). This value is considerably lower than that found with reovirus (Shatkin and Banerjee, 1970) or phage RF-RNA (Bishop and Levintow, 1971). Part of the reannealed poliovirus RNA is found in a structure resembhng RI-RNA (Bishop and Levintow, 1971) (Koch and Vollertsen, unpubhshed). Therefore, the estimate of the synthesis of pohovirus-specific RNA in E. coli determined by RNase resistance of the annealed RNA represents only a minimal... 

In other experiments, poliovirus minus-strand RNA was used to detect newly synthesized viral plus-strand RNA. The extent of conversion of newly synthesized RNA from RNase-sensitive to RNase-resistant form was again found to depend on the relative proportions of the two RNAs. However, the... 

The experimental results described above leave one important question unanswered, namely What is the mechanism of RNA synthesis in RF-RNA-infected F. coli Does the RF-RNA serve as a template for an E. coli enzyme directing the synthesis of new virus-specific RNA — both virus plus- and minus-strand RNA — or is the adsorbed RF-RNA melted in E. coli, with the result that its viral plus-strand in turn serves as mRNA for the synthesis of a virus-specific polymerase responsible for the subsequent accumulation of new virus-specific RNA We attempted to answer this question by studying the fate of viral RNA in E. coli incubated in the absence and presence of inhibitors of protein synthesis, using the methods outlined above. 

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