In vitro transcription of 70S RNA by the RNA-directed DNA polymerase of Rouse sarcoma virus: lack of influence of RNase H.

The influence of Rous sarcoma virus (RSV)-associated RNase H on the in vitro synthesis of DNA by the RSV RNA-directed DNA polymerase was determined under conditions whereby RNase H activity was selectively inhibited with NaF. Not only were we unable to detect any effect on the size, structure, or genetic complixity of the DNA product synthesized in the absence of RNase H activity, but the displacement of DNA from the 70S RNA:DNA hybrid structures was also unaffected. The suitability of 70S RNA:DNA hybrid structures synthesized in vitro to serve as a substrate for RNase H is discussed.     

Rous sarcoma virus nucleic acid-binding protein p12 is necessary for viral 70S RNA dimer formation and packaging.

To study the function(s) of the Rous sarcoma virus nucleic acid-binding protein p12, we constructed mutants by using two restriction sites in the p12 proviral coding sequence of the Prague C strain to insert KpnI synthetic linkers. The two restriction sites are in the same reading frame, which allowed us to construct a deletion mutant lacking the two conserved Cys-His regions and a duplication mutant containing three intact Cys-His boxes. These mutant DNAs were transfected into chicken embryo fibroblasts, and the viral particles produced in a transient assay were characterized biochemically and for infectivity. Our results indicate that the Rous sarcoma virus nucleic acid-binding protein p12 is necessary for genomic RNA packaging but not for particle assembly and is implicated in the formation of a stable 70S dimeric RNA. Moreover, the fact that one mutant was apparently able to package normal 70S RNA but was not infectious suggests a role for p12 during the infection process.     

Nucleotide sequence of Rous sarcoma virus RNA at the initiation site of DNA synthesis: The 102nd nucleotide is U.

The T₁ oligonucleotide in the genome Rous sarcoma virus (RSV) that corresponds to the initiation site of DNA synthesis in vitro was identified by hybridization of genome RNA with RSV strong stop DNA (the initial 101-nucleotide long fragment synthesized in endogenous reactions) and partially sequenced. The sequence of (C₂, U₂) A-U-U-U-G found corresponds to the d(A-A-T-G-A-A-G) sequence at the 5′ end of the DNA product plus the CA-OH sequence at the 3′ end of the tRNA^Trp primer. Therefore the nucleotide opposite the terminal A of the primer is the complementary U. Furthermore, no internal repetition of more than 30 nucleotides of the 5′ sequence could be detected.     

Control of template positioning during de novo initiation of RNA synthesis by the bovine viral diarrhea virus NS5B polymerase

The RNA-dependent RNA polymerase of the hepatitis C virus and the bovine viral diarrhea virus(BVDV)is able to initiate RNA synthesis denovo in the absence of a primer. Previous crystallographic data have pointed to the existence of a GTP-specific binding site (G-site) that is located in the vicinity of the active site of the BVDV enzyme. Here we have studied the functional role of the G-site and present evidence to show that specific GTP binding affects the positioning of the template during de novo initiation. Following the formation of the first phosphodiester bond, the polymerase translocates relative to the newly synthesized dinucleotide, which brings the 5'-end of the primer into the G-site, releasing the previously bound GTP. At this stage, the 3'-end of the template can remain opposite to the 5'-end of the primer or be repositioned to its original location before RNA synthesis proceeds. We show that the template can freely move between the two locations, and both complexes can isomerize to equilibrium. These data suggest that the bound GTP can stabilize the interaction between the 3'-end of the template and the priming nucleotide, preventing the template to overshoot and extend beyond the active site during de novo initiation. The hepatitis C virus enzyme utilizes a dinucleotide primer exclusively from the blunt end; the existence of a functionally equivalent G-site is therefore uncertain. For the BVDV polymerase we showed that de novo initiation is severely compromised by the T320A mutant that likely affects hydrogen bonding between the G-site and the guanine base. Dinucleotide-primed reactions are not influenced by this mutation, which supports the notion that the G-site is located in close proximity but not at the active site of the enzyme.     

Template requirement and initiation site selection by hepatitis C virus polymerase on a minimal viral RNA template

RNA-dependent RNA polymerase, NS5B protein, catalyzes replication of viral genomic RNA, which presumably initiates from the 3'-end. We have previously shown that NS5B can utilize the 3'-end 98-nucleotide (nt) X region of the hepatitis C virus (HCV) genome as a minimal authentic template. In this study, we used this RNA to characterize the mechanism of RNA synthesis by the recombinant NS5B. We first showed that NS5B formed a complex with the 3'-end of HCV RNA by binding to both the poly(U-U/C)-rich and X regions of the 3'-untranslated region as well as part of the NS5B-coding sequences. Within the X region, NS5B bound stem II and the single-stranded region connecting stem-loops I and II. Truncation of 40 nt or more from the 3'-end of the X region abolished its template activity, whereas X RNA lacking 35 nt or less from the 3'-end retained template activity, consistent with the NS5B-binding site mapped. Furthermore, NS5B initiated RNA synthesis from a specific site within the single-stranded loop I. All of the RNA templates that have a double-stranded stem at the 3'-end had the same RNA initiation site. However, the addition of single-stranded nucleotides to the 3'-end of X RNA or removal of double-stranded structure in stem I generated RNA products of template size. These results indicate that HCV NS5B initiates RNA synthesis from a single-stranded region closest to the 3'-end of the X region. These results have implications for the mechanism of HCV RNA replication and the nature of HCV RNA templates in the infected cells.     

Polyamines stimulate natural RNA-directed DNA synthesis by Rauscher murine leukemia virus DNA polymerase

In the presence of optimal concentrations of Mg²⁺, spermine and spermidine were found to stimulate rabbit globin mRNA-directed cDNA synthesis by Rauscher murine leukemia virus (R-MuLV) DNA polymerase. Stimulation of DNA synthesis did not occur with the polyamines putrescine or cadaverine, nor could exogenously provided salt or ammonium ions duplicate the stimulation. Analysis of the mechanism of stimulation showed that inclusion of spermine in reaction mixtures a) increased Vmax and decreased apparent Km with respect to the globin mRNA-oligo(dT) tem?late-primer complex, and b) decreased the quantity of oligo (dT) required for optimal rates of cDNA synthesis on a fixed quantity of mRNA template. Genomic 70S RNA-directed cDNA synthesis was also stimulated by spermine addition to reaction mixtures, but only at supra-optimal RNA concentrations. Our results suggest that stimulation of R-MuLV DNA polymerase activity by polyamines is primarily due to stabilization of the enzyme-templateprimer initiation complex resulting in increased efficiency of initiation of cDNA synthesis.     

Regulation of Rous sarcoma virus RNA-dependent DNA polymerase isoenzymes by in vitro phosphorylation-dephosphorylation

The activity of the αβ form of Rous sarcoma virus RNA-dependent DNA polymerase was stimulated upon treatment with the protein kinase purified from the same virus. This enhancement was observed for both DNA-dependent and RNA-dependent DNA polymerase activities, whereas the RNase H activity associated with the polymerase was not affected. On the other hand, the protein kinase did not induce detectable changes in the activities of the α-polymerase isoenzyme. Treatment with Escherichia coli alkaline phosphatase resulted in a reduction of the polymerase activities of the αβ isoenzyme with no effects on RNase H as well as on the α form of the DNA polymerase. Preincubations of the αβ- and α-oncornaviral polymerase isoenzymes with two other protein kinases—from avian myeloblastosis virus and from beef heart (catalytic subunit)—had no substantial effects on DNA polymerase and RNase H activities of both polymerase isoenzymes. Both α and β subunits of the polymerase isoenzymes were phosphorylated in vitro by all three protein kinases employed, although only the β subunit was shown previously to be phosphorylated in vivo.     

Viral DNA synthesis defects in assembly-competent Rous sarcoma virus CA mutants

The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle.     

Initiation of DNA synthesis by the avian oncornavirus RNA-directed DNA polymerase: tryptophan tRNA as the major species of primer RNA.

The major species of primer RNA required for the initiation of DNA synthesis by the Rous sarcoma virus RNA-directed DNA polymerase can be aminoacylated by tryptophan. Furthermore, an intact 3' terminus is required for the primer to function in the initiation of DNA synthesis.     

Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase

The minimal RNA synthesis machinery of non-segmented negative-strand RNA viruses comprises a genomic RNA encased within a nucleocapsid protein (N-RNA), and associated with the RNA-dependent RNA polymerase (RdRP). The RdRP is contained within a viral large (L) protein, which associates with N-RNA through a phosphoprotein (P). Here, we define that vesicular stomatitis virus L initiates synthesis via a de-novo mechanism that does not require N or P, but depends on a high concentration of the first two nucleotides and specific template requirements. Purified L copies a template devoid of N, and P stimulates L initiation and processivity. Full processivity of the polymerase requires the template-associated N protein. This work provides new mechanistic insights into the workings of a minimal RNA synthesis machine shared by a broad group of important human, animal and plant pathogens, and defines a mechanism by which specific inhibitors of RNA synthesis function.     

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.