Effects of Mutations in the G Tract of the Human Immunodeficiency Virus Type 1 Polypurine Tract on Virus Replication and RNase H Cleavage

Julias, John G.; McWilliams, Mary Jane; Sarafianos, Stefan G.; Alvord, W. Gregory; Arnold, Edward; Hughes, Stephen H.

doi:10.1128/jvi.78.23.13315-13324.2004

Cited by 34 publications

(26 citation statements)

References 37 publications

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“…This viral RNA then is used both as the viral genome and as viral mRNA (2,8,9,17,27,30). In the integration step, IN recognizes sequences at the ends of linear viral DNA, termed the attachment site (att), and usually removes two nucleotides adjacent to the conserved CA dinucleotide on each of the 3Ј ends of the linear viral DNA (6,11,15,28). In the next step, the processed ends of the viral DNAs are joined to the host chromosomal DNA.…”

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confidence: 99%

Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

Chang

Wierzchoslawski

et al. 2008

J Virol

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The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3 end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5 end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5 end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo.The retroviral life cycle is characterized by the conversion of the single-stranded RNA genome found in virions into a double-stranded linear DNA that is subsequently integrated into the host cell genome. Viral DNA synthesis takes place in the cytoplasm of the infected cell. The RNA genome of the virus is copied into DNA by a virally encoded enzyme, reverse transcriptase (RT). RT contains two enzymatic activities, a DNA polymerase that can copy either an RNA or a DNA template, and an RNase H that cleaves RNA only if the RNA is part of an RNA/DNA hybrid. Like many other DNA polymerases, RT requires both a template and a primer. First (minus)-strand DNA synthesis is initiated from a cellular tRNA primer that is base paired at the primer binding site (PBS), which is near the 5Ј end of the viral genomic RNA. During the reverse transcription process, this tRNA primer is removed from the end of the minus-strand DNA by RNase H; this defines the right (U5) end of the linear viral DNA. Second (plus)-strand DNA synthesis is initiated from a polypurine tract (PPT) primer adjacent to U3. The RNase H cleavages that generate and remove PPT primer define the left (

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Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

Chang

Wierzchoslawski

et al. 2008

J Virol

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“…A virally encoded integrase (IN) recognizes sequences at the ends of the linear molecule and catalyzes integration in a two-step reaction. In the first step, integrase removes a specific number of nucleotides, usually two, from each of the 3Ј ends of the linear viral DNA (6,9,16,27). The processed ends are then joined to host chromosomal DNA by integrase, creating a duplication of a short sequence from the target site, which flanks the integrated provirus as a direct repeat of 4 to 6 bp (8,14,15,27).…”

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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

Chang

Hughes

2006

J Virol

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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.

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“…Produce VSV-g-pseudotyped HIV by transfecting 293 cells (as shown in Figure 1, step 1) [32][33][34] .…”

Section: Generation Of Virus Stocksmentioning

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Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Smith

Hughes

2014

JoVE

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Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

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Effects of Mutations in the G Tract of the Human Immunodeficiency Virus Type 1 Polypurine Tract on Virus Replication and RNase H Cleavage

Cited by 34 publications

References 37 publications

Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

Rous Sarcoma Virus (RSV) Integration In Vivo: a CA Dinucleotide Is Not Required in U3, and RSV Linear DNA Does Not Autointegrate

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

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

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