Viral Plus strand

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. 

The direct visualization of poliovirus NA in living host cells was achieved with MB, involving TAMRA as F and Dabcyl as Q, targeting the viral plus-strand RNA (25). The poliovirus plus strand was observed to display different distribution patterns at different post-infection time points in living vero cells. 

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. 

As noted, the viral RNA is of the plus (+) sense. Replicase synthesizes RNA of minus (-) sense using the infecting RNA as template. After minus RNA has been synthesized, plus RNA is made from this minus RNA. The newly made plus RNA strands now serve as messengers for virus protein synthesis. The gene for the maturation protein is at the 5 end of the RNA. Translation of the gene coding for the maturation protein (needed in only one copy per virus particle), occurs only from the newly formed plus-strand RNA as... 

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)...

HIV-1 RT contains RNase H, which is responsible for degradation of viral RNA and removal of RNA primers for minus- and plus-strand DNA synthesis (see reviews [87-89]). The absolute requirement for virus-associated RNase H function [90-93] offers an additional target for antiretroviral drugs. The RNase H domain of HIV-1 RT is located at the C-terminus of the p66 subunit (Figures 2 and 3). In contrast to the polymerase domain of HIV-1 RT, the structure of the RNase H domain is quite similar in all known HIV-1 RT structures and conforms quite well with the structure of the isolated HIV-1 RNase H domain [94-95]. The relative stability of the structure of the RNase H domain suggests that the RNase H active site could be a relatively well-defined target for drug... 

Following viral infection the virus-plus strand first functions as an mRNA for the synthesis of the viral proteins. Once the viral reverse transcriptase has been synthe-... 

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. 

Basically, in the absence of a better template, the viral replicase would bind to, and initiate the transcription of, the viral RNA. Conceivably, the very first step of this process would be the synthesis of the poly(U) tract (Figure 4> A), followed by the transcription of the heteropolymeric portion of the "plus" strand (Figure 4> But as soon as the first complementary strand is... 

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

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. 

Assuming that the leader gene is the site of entry for the VSV polymerases, it has been proposed that the VSV leader RNA sequence regulates the switch from transcription to replication (Blum-berg et al., 1981,1983). This led Kolakofsky and co-workers to speculate that the switch involves the leader sequence and the specific concentration of N protein. Wilusz et al. (1984) have proposed that formation of an La-protein-leader RNA complex may serve as an in vivo attenuator of transcription to ensure adequate levels of viral protein accumulation before the switch to replication takes place. Evidence for displacement of La protein by viral N protein on the plus-strand leader RNA is consistent with this possibility (Kurilla and Keene, 1984). 

The major products of VSV transcription are complementary RNA molecules, either full-length templates to replicate progeny negative strands, or monocistronic messengers for each of the five viral genes preceded by a plus-strand uncapped and nonpolyadenylated... 

We have not yet been able to identify the cellular target for VSV inhibition of DNA synthesis, but studies summarized here rule out a viral effect on nucleoside transport, DNA degradation, inactivation of DNA polymerase and thymidine kinase, or premature termination of already initiated DNA chains. By exclusion, we hypothesize that VSV inhibits DNA synthesis by blocking initiation of DNA replication, perhaps in a manner similar to its inhibition of RNA transcription (McGowan et al., 1982). The ambiguities inherent in the in vitro DNA polymerase assay make it difficult to rule out conclusively cellular enzymes as targets for VSV inhibition of DNA replication. Further studies are necessary to test the hypothesis that a VSV product, such as the plus-strand leader RNA, inhibits initiation of both RNA transcription and DNA replication. 

In nature QB and MS2 infect bacteria like Escherichia coli. The life cycle of the virus consists of a regulated sequence of processes (Fig. 6) (1) translation of viral RNA by the host s machinery yielding the virus dependent subunit of the specific RNA replicase, (2) RNA replication by the specific enzyme yielding minus- and lateron plus-strands, (3) translation of viral RNA leading to A-protein, coat protein and lysis protein, (4) formation of virus particles and (5) lysis of the bacterial cell. 

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