Structure of murine sarcoma virus DNA replicative intermediates synthesized in vitro

We have established an assay for the function of preintegration complexes (PICs) of human immunodeficiency virus type 2 (HIV-2) to investigate the integration mechanism and to develop additional methods for screening candidate integration inhibitors. We partially purified HIV-2 PICs and found that they were competent to integrate viral cDNA into target DNA in vitro. Analysis of the structure of integration products on Southern blots revealed forms consistent with those expected for authentic integration products and circular forms containing one and two long terminal repeats. To determine whether in vitro products had the detailed structure expected of integration products formed in vivo, we recovered product molecules and analyzed junctions between viral DNA and target DNA. In the integration junctions of all nine molecules examined, we observed the 5-bp duplication of target sequence characteristic of integration in vivo. We investigated the possible role in integration of Vpx, a protein present in HIV-2 but not HIV-1 and known to be present in viral cores. Although association of Vpx with viral cDNA was detectable, our studies revealed no obvious role of Vpx in integration since the activities of PICs from Vpx ؊ virions were indistinguishable from those of wild type. We have also investigated the use of HIV-2 PICs as tools to screen candidate HIV inhibitors. Assays with HIV-2 PICs, like assays with HIV-1 PICs, were less sensitive to many small molecule inhibitors than were reactions with purified integrase only. Comparing results of assays with PICs from HIV-1 and HIV-2 may be particularly useful, since inhibitors active against both may be more widely useful and less vulnerable to escape mutants.

supporting

confidence: 64%

Human immunodeficiency virus type 2 preintegration complexes: activities in vitro and response to inhibitors

Hansen

Bushman

1997

“…precursors to the full-length plus strands, as suggested in the models proposed by Gilboa et al (7) and Dina and Benz for MuLV DNA (4). DISCUSSION Sizes of plus strands of linear retroviral DNA.…”

Section: ' Gpmentioning

confidence: 85%

“…300 nucleotides composed of sequences from both ends of viral RNA (11,23): the early plus strand might serve as the template for the synthesis of the 3' end of the minus strand, thereby producing the terminal redundancy (1,23,32). This notion has gained considerable support from the finding of plus strands equal in size and genetic content to the terminal redundancy of murine leukemia virus (MuLV) linear DNA after synthesis of MuLV DNA in detergent-disrupted virions (4,7,15).…”

mentioning

confidence: 99%

Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus

Kung

Fung

Majors

et al. 1981

The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DNA (0.35 kb), and it annealed with complementary DNAs specific for the 3' and 5' termini of viral RNA (Varmus et al., J. Mol. Biol. 120:50-82, 1978). Several subgenomic plus-strand fragments (0.94, 1.38, 2.3, and 3.4 kb) annealed with these reagents. At least the 0.94-and 1.38-kb strands were located at the same end of linear DNA as the 0.35-kb strand, indicating that multiple specific sites for initiation were employed to generate strands which overlapped on the structural map. We were unable to detect RNA linked to plus strands isolated as early as 2.5 h postinfection; thus, the primers must be short (fewer than 50 to 100 nucleotides), rapidly removed, or not composed of RNA. To determine whether multiple priming events are a general property of retroviral DNA synthesis in vivo, we also examined plus strands of mouse mammary tumor virus DNA in chronically infected rat cells after induction of RNA and subsequent

“…The circularization of purified linear viral DNA in extracts from uninfected cells provides strong evidence that 1-LTR circles are the result of host-mediated recombination between the LTRs on linear viral DNA. Alternative models, based on circular DNA molecules produced during the reverse transcription of viral RNA in permeabilized virions, have been proposed to account for the formation of 1-LTR circles (7,14). In these models, 1-LTR circles result from a circular replication intermediate that results from the failure of reverse transcriptase to catalyze displacement synthesis of the nascent viral DNA through the LTR region.…”

Section: Discussionmentioning

confidence: 99%

“…While it has been demonstrated that the linear viral DNA synthesized in the cytoplasm is the precursor to the circular DNA forms found in the nucleus (25), the mechanism of circularization remains unknown. Circles containing one LTR have been proposed to arise either from homologous recombination between the LTRs present on the linear viral DNA molecule (13,24) or from a circular intermediate in the formation of the linear molecule during reverse transcription (7,14). Circles with two tandem LTRs, on the other hand, are presumed to arise by the direct ligation of the ends of the linear DNA molecule (15,29).…”

mentioning

confidence: 99%

Circularization of human immunodeficiency virus type 1 DNA in vitro

Farnet

Haseltine

1991

133

Linear viral DNA present in cytoplasmic extracts of cells newly infected with human immunodeficiency virus type 1 can be induced to form 1-LTR and 2-LTR circles by incubation of the extracts in the presence of added nucleoside triphosphates. No circular DNA forms are detected when extracts are incubated in the absence of added nucleoside triphosphates. Restriction enzyme analysis and polymerase chain reaction analysis with selected primers, as well as DNA sequence analysis of the polymerase chain reaction products, show that most of the 2-LTR circles are the result of autointegration reactions, while 1-LTR circles result from recombination between the long terminal repeats on the linear viral DNA. In addition, a small amount of simple 2-LTR circles, formed by end-to-end joining of the linear viral DNA, is formed in vitro. Integration of the linear viral DNA into heterologous DNA competes effectively with the formation of 2-LTR circles by autointegration. However, concentrations of target DNA which completely block autointegration have no effect on the formation of 1-LTR circles or simple 2-LTR circles. Factors present in extracts of uninfected cells can mediate the formation of 1-LTR circles and simple 2-LTR circles from purified deproteinated linear viral DNA, indicating that viral proteins are not necessary for the formation of these two types of circular viral DNA. These experiments demonstrate that all the transformations of linear viral DNA which occur in the nuclei of cells infected with human immunodeficiency virus type 1 can be reproduced in vitro.