Somatic cell mutants resistant to retrovirus replication: intracellular blocks during the early stages of infection - PubMed (original) (raw)

Somatic cell mutants resistant to retrovirus replication: intracellular blocks during the early stages of infection

G Gao et al. Mol Biol Cell. 1999 Jun.

Free PMC article

Abstract

To identify cellular functions involved in the early phase of the retroviral life cycle, somatic cell mutants were isolated after selection for resistance to infection. Rat2 fibroblasts were treated with chemical mutagens, and individual virus-resistant clones were recovered after selection for resistance to infection. Two clones were characterized in detail. Both mutant lines were resistant to infection by both ecotropic and amphotropic murine viruses, as well as by human immunodeficiency virus type 1 pseudotypes. One clone showed a strong block to reverse transcription of the retroviral RNA, including formation of the earliest DNA products. The second clone showed normal levels of viral DNA synthesis but did not allow formation of the circular DNAs normally found in the nucleus. Cell fractionation showed that the viral preintegration complex was present in a form that could not be extracted under conditions that readily extracted the complex from wild-type cells. The results suggest that the DNA was trapped in a nonproductive state and excluded from the nucleus of the infected cell. The properties of these two mutant lines suggest that host gene products play important roles both before and after reverse transcription.

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Figures

Figure 1

Figure 1

Schematic representation of the strategy for isolating mutant cells resistant to retroviral infection. TK−, thymidine kinase deficient; TK+, thymidine kinase proficient.

Figure 2

Figure 2

Resistance of mutant cells to GFP virus infection. RC-2 cells (open triangles), R3-2 cells (closed circles), and R4-7 cells (open circles) were infected with the indicated viruses at indicated dilution. The number of infected cells, as indicated by expression of GFP, was determined by flow cytometry.

Figure 3

Figure 3

Analysis of viral DNA in infected mutant cells. RC-2 and mutant cells were infected with various dilutions of virus, and the amount of viral DNA synthesized in the infected cells was measured by PCR under conditions controlled such that the yield of PCR product was proportional to the input DNA. (A) Cells were infected with MuLV-GFP virus at various dilutions. At different time points the viral DNA in the infected cells was extracted and detected by PCR using primers that specifically amplify the GFP sequence. (B) The same preparations at the 24-h postinfection time point in A were used as templates to detect the synthesis of plus strand viral DNA in the infected cells. The migration position of the PCR product is indicated. (C) Cells were infected with either ecotropic MuLV-GFP or ecotropic MuLV-N2 virus at various dilutions, as indicated. Twenty four hours after infection the cells were collected, and minus strand strong stop DNA was detected by PCR. The migration position of the PCR product is indicated. (D) RC-2 and R4-7 cells were infected with amphotropic MuLV-GFP virus at various dilutions. Twenty-four hours after infection, viral DNA synthesis was analyzed by PCR using primers that amplify GFP sequences. (E) Cells were infected with VSV-G–pseudotyped HIV-GFP virus at various dilutions, as indicated. At 24 h after infection, cells were collected, and minus strand strong stop DNA and plus strand DNA were detected by PCR. The migration positions of the PCR products are indicated. H.I. virus, heat-inactivated virus was used as a control for plasmid DNA contamination of the virus. (F) The same preparations from A were analyzed by PCR using primers that specifically amplify the LTR–LTR junction to detect circular DNAs in the nucleus.

Figure 4

Figure 4

Schematic representation of the procedure for fractionating infected cells. RC-2 and R3-2 cells were infected with undiluted ecotropic MuLV-GFP virus and extracted with either hypotonic buffer (A) or buffer containing digitonin (B).

Figure 5

Figure 5

Analysis of viral DNA in the fractionated cell extracts by PCR using primers that amplify GFP sequences. The migration positions of the PCR product are indicated with arrows. (A) Four or 8 h after infection cells were collected and fractionated. The low-molecular-weight DNA in the pellet (Hirt extract) was resuspended in 20 μl of Tris-EDTA plus 10 μg/ml RNase A. Five of 300 μl of cytoplasmic extract or 1 of 20 μl of the pellet DNA were PCR amplified to detect viral DNA. (B) Eight hours after infection the cells were collected by trypsinization. One-third of the cells were used to extract total DNA by the Hirt procedure. The rest of the cells were fractionated as described in Figure 4B. Five of 300 μl of each extract or 1 of 60 μl of the total DNA were used to assay viral DNA by PCR. (C) The cytoplasmic extracts from B were fractionated by centrifugation on a 20–70% sucrose gradient. The gradient was equally divided into 10 fractions of 500 μl each. Five microliters of solution from each fraction were used to detect viral DNA by PCR.

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