scholarly journals Soluble Rous Sarcoma Virus Reverse Transcriptases α, αβ, and β Purified from Insect Cells Are Processive DNA Polymerases That Lack an RNase H 3′ → 5′ Directed Processing Activity

1999 ◽  
Vol 274 (37) ◽  
pp. 26329-26336 ◽  
Author(s):  
Susanne Werner ◽  
Birgitta M. Wöhrl
Keyword(s):  
Rous Sarcoma Virus ◽  
Insect Cells ◽  
Dna Polymerases ◽  
Rnase H ◽  
Reverse Transcriptases ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Processing Activity

scholarly journals Requirements for Minus-Strand Transfer Catalyzed by Rous Sarcoma Virus Reverse Transcriptase

2001 ◽  
Vol 75 (21) ◽  
pp. 10132-10138 ◽  
Author(s):  
Susanne Werner ◽  
Karin Vogel-Bachmayr ◽  
Britta Hollinderbäumer ◽  
Birgitta M. Wöhrl
Keyword(s):  
Rous Sarcoma Virus ◽  
Rnase H ◽  
Strand Transfer ◽  
Minus Strand ◽  
Reverse Transcriptases ◽  
Rous Sarcoma ◽  
Immunodeficiency Virus ◽  
Different Types ◽  
Sarcoma Virus ◽  
Template Substrate

ABSTRACT We have examined the specific minus-strand transfer reactions that occur after the synthesis of minus strong-stop DNA and nonspecific strand switching on homopolymeric poly(rA) templates with different types of Rous sarcoma virus (RSV) reverse transcriptases. Three different types of reverse transcriptases can be isolated from virions of RSV: heterodimeric αβ and homodimeric α and β. The mechanism of minus-strand transfer was examined using a model primer-template substrate corresponding to the 5′- and 3′-terminal RNA regions of the RSV genome. The results reveal that the RNase H activity of RSV reverse transcriptases is required for minus-strand transfer. Less than 2% of strand transfer of the extended product is detectable with RNase H-deficient enzymes. We could show that the α homodimer lacking the integrase domain can perform strand transfer almost as efficiently as the αβ and αPol heterodimers. In contrast, the activities of β and Pol for minus-strand transfer are reduced. Furthermore, a two- to fivefold increase in minus-strand transfer activities was observed in the presence of human immunodeficiency virus type 1 nucleocapsid protein.

scholarly journals Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

2005 ◽  
Vol 79 (21) ◽  
pp. 13694-13704 ◽  
Author(s):  
Kevin W. Chang ◽  
John G. Julias ◽  
W. Gregory Alvord ◽  
Jangsuk Oh ◽  
Stephen H. Hughes
Keyword(s):  
Rous Sarcoma Virus ◽  
Leukemia Virus ◽  
Shuttle Vector ◽  
Rnase H ◽  
Wild Type Virus ◽  
Reverse Transcriptases ◽  
Preferential Cleavage ◽  
Rous Sarcoma ◽  
Duck Hepatitis ◽  
Sarcoma Virus

ABSTRACT Retroviral polypurine tracts (PPTs) serve as primers for plus-strand DNA synthesis during reverse transcription. The generation and removal of the PPT primer requires specific cleavages by the RNase H activity of reverse transcriptases; removal of the PPT primer defines the left end of the linear viral DNA. We replaced the endogenous PPT from RSVP(A)Z, a replication-competent shuttle vector based on Rous sarcoma virus (RSV), with alternate retroviral PPTs and the duck hepatitis B virus “PPT.” Viruses in which the endogenous RSV PPT was replaced with alternate PPTs had lower relative titers than the wild-type virus. 2-LTR circle junction analysis showed that the alternate PPTs caused significant decreases in the fraction of viral DNAs with complete (consensus) ends and significant increases in the insertion of part or all of the PPT at the 2-LTR circle junctions. The last two nucleotides in the 3′ end of the RSV PPT are GA. Examination of the (mis)cleavages of the alternate PPTs revealed preferential cleavages after GA dinucleotide sequences. Replacement of the terminal 3′ A of the RSV PPT with G caused a preferential miscleavage at a GA sequence spanning the PPT-U3 boundary, resulting in the deletion of the terminal adenine normally present at the 5′ end of the U3. A reciprocal G-to-A substitution at the 3′ end of the murine leukemia virus PPT increased the relative titer of the chimeric RSV-based virus and the fraction of consensus 2-LTR circle junctions.

scholarly journals Asymmetric Subunit Organization of Heterodimeric Rous Sarcoma Virus Reverse Transcriptase αβ: Localization of the Polymerase and RNase H Active Sites in the α Subunit

2000 ◽  
Vol 74 (7) ◽  
pp. 3245-3252 ◽  
Author(s):  
Susanne Werner ◽  
Birgitta M. Wöhrl
Keyword(s):  
Reverse Transcriptase ◽  
Active Site ◽  
Rous Sarcoma Virus ◽  
Active Sites ◽  
Polymerase Activity ◽  
Rnase H ◽  
Wild Type ◽  
Α Subunit ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT The genes encoding the α (63-kDa) and β (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV αβ and αPol RTs within which one subunit was selectively mutated. When αβ heterodimers contained the Asp 181→Asn mutation in their β subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their α subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in αβ heterodimers mutated in the β subunit but was lost when the mutation was present in the α subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the α subunit of the heterodimeric RSV RT αβ.

scholarly journals The Effects of Alternate Polypurine Tracts (PPTs) and Mutations of Sequences Adjacent to the PPT on Viral Replication and Cleavage Specificity of the Rous Sarcoma Virus Reverse Transcriptase

2008 ◽  
Vol 82 (17) ◽  
pp. 8592-8604 ◽  
Author(s):  
Kevin W. Chang ◽  
Jangsuk Oh ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Reverse Transcriptase ◽  
Rous Sarcoma Virus ◽  
Point Mutations ◽  
Rnase H ◽  
Wild Type ◽  
Viral Dna ◽  
Specific Sequence ◽  
Cleavage Specificity ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT We previously reported that a mutant Rous sarcoma virus (RSV) with an alternate polypurine tract (PPT), DuckHepBFlipPPT, had unexpectedly high titers and that the PPT was miscleaved primarily at one position following a GA dinucleotide by the RNase H of reverse transcriptase (RT). This miscleavage resulted in a portion of the 3′ end of the PPT (5′-ATGTA) being added to the end of U3 of the linear viral DNA. To better understand the RNase H cleavage by RSV RT, we made a number of mutations within the DuckHepBFlipPPT and in the sequences adjacent to the PPT. Deleting the entire ATGTA sequence from the DuckHepBFlipPPT increased the relative titer to wild-type levels, while point mutations within the ATGTA sequence reduced the relative titer but had minimal effects on the cleavage specificity. However, mutating a sequence 5′ of ATGTA affected the relative titer of the virus and caused the RNase H of RSV RT to lose the ability to cleave the PPT specifically. In addition, although mutations in the conserved stretch of thymidine residues upstream of the PPT did not affect the relative titer or cleavage specificity, the mutation of some of the nucleotides immediately upstream of the PPT did affect the titer and cleavage specificity. Taken together, our studies show that the structure of the PPT in the context of the cognate RT, rather than a specific sequence, is important for the proper cleavage by RSV RT.

DNA Polymerases of Rous Sarcoma Virus: Delineation of Two Reactions with Actinomycin

Nature ◽  
1970 ◽  
Vol 228 (5270) ◽  
pp. 433-435 ◽  
Author(s):  
JEROME P. MCDONNELL ◽  
AXEL-CLAUDE GARAPIN ◽  
WARREN E. LEVINSON ◽  
NANCY QUINTRELL ◽  
LOIS FANSHIER ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Dna Polymerases ◽  
Rous Sarcoma ◽  
Sarcoma Virus

scholarly journals Mutations in the U5 Sequences Adjacent to the Primer Binding Site Do Not Affect tRNA Cleavage by Rous Sarcoma Virus RNase H but Do Cause Aberrant Integrations In Vivo

2006 ◽  
Vol 80 (1) ◽  
pp. 451-459 ◽  
Author(s):  
Jangsuk Oh ◽  
Kevin W. Chang ◽  
Stephen H. Hughes
Keyword(s):  
Binding Site ◽  
Rous Sarcoma Virus ◽  
Virus Titer ◽  
Rnase H ◽  
Primer Binding Site ◽  
Rous Sarcoma ◽  
Trna Primer ◽  
Sarcoma Virus ◽  
The Right ◽  
Hiv 1

ABSTRACT In most retroviruses, the first nucleotide added to the tRNA primer becomes the right end of the U5 region in the right long terminal repeat (LTR); the removal of this tRNA primer by RNase H defines the right end of the linear double-stranded DNA. Most retroviruses have two nucleotides between the 5′ end of the primer binding site (PBS) and the CA dinucleotide that will become the end of the integrated provirus. However, human immunodeficiency virus type 1 (HIV-1) has only one nucleotide at this position, and HIV-2 has three nucleotides. We changed the two nucleotides (TT) between the PBS and the CA dinucleotide of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z to match the HIV-1 sequence (G) and the HIV-2 sequence (GGT), and we changed the CA dinucleotide to TC. In all three mutants, RNase H removes the entire tRNA primer. Sequence analysis of RSVP(HIV2) proviruses suggests that RSV integrase can remove three nucleotides from the U5 LTR terminus of the linear viral DNA during integration, although this mutation significantly reduced virus titer, suggesting that removing three nucleotides is inefficient. However, the results obtained with RSVP(HIV1) and RSVP(CATC) show that RSV integrase can process and integrate the normal U3 LTR terminus of a linear DNA independently of an aberrant U5 LTR terminus. The aberrant end can then be joined to the host DNA by unusual processes that do not involve the conserved CA dinucleotide. These unusual events generate either large duplications or, less frequently, deletions in the host genomic DNA instead of the normal 5- to 6-base duplications.

scholarly journals A Substitution in Rous Sarcoma Virus Integrase That Separates Its Two Biologically Relevant Enzymatic Activities

2005 ◽  
Vol 79 (8) ◽  
pp. 4691-4699 ◽  
Author(s):  
Wesley M. Konsavage ◽  
Stephen Burkholder ◽  
Malgorzata Sudol ◽  
Amy L. Harper ◽  
Michael Katzman
Keyword(s):  
Rous Sarcoma Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Biologically Relevant ◽  
Rous Sarcoma ◽  
Unintegrated Dna ◽  
Sarcoma Virus ◽  
Processing Activity ◽  
Retroviral Integrase ◽  
Dna Ends

ABSTRACT Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant “−2” site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of −2 nicks to −3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.

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