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. 2001 Jun;75(11):5433-40.
doi: 10.1128/JVI.75.11.5433-5440.2001.

Feline immunodeficiency virus cell entry

S C Frey  1 , E A Hoover, J I Mullins
Affiliations

Feline immunodeficiency virus cell entry

S C Frey et al. J Virol. 2001 Jun.

Abstract

The process of feline immunodeficiency virus (FIV) cell entry was examined using assays for virus replication intermediates. FIV subtype B was found to utilize the chemokine receptor CXCR4, but not CCR5, as a cellular receptor. Zidovudine blocked formation of late viral replication products most effectively, including circular DNA genome intermediates. Our findings extend the role of CXCR4 as a primary receptor for CD4-independent cell entry by FIV.

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Figures

FIG. 1
FIG. 1
CrFK cell infection with FIV 34TF10 and detection of viral replication intermediates. (A) CrFK cells were infected with FIV 34TF10 in replicate wells and harvested from one well at 0, 6, 20, 30, 45, and 70 h PI. PCR was performed as described in the text, with FIV 2542-CRFK cellular DNA (FIV+) as the positive control and H2O plus reagents (H2O) as the negative control. The DNA marker is a 100-bp or 1-kb ladder, as indicated. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (B) Schematic representation of primer positions for derivation of PCR products in FIV-infected cells derived from linear and circular viral DNA is shown. (C) One-LTR circle junction fragment homology (shaded segments) is indicated. As shown at the bottom of panel C, the fragments produced after FIV 34TF10 infection were colinear with that expected from the FIV 34TF10 genome.
FIG. 1
FIG. 1
CrFK cell infection with FIV 34TF10 and detection of viral replication intermediates. (A) CrFK cells were infected with FIV 34TF10 in replicate wells and harvested from one well at 0, 6, 20, 30, 45, and 70 h PI. PCR was performed as described in the text, with FIV 2542-CRFK cellular DNA (FIV+) as the positive control and H2O plus reagents (H2O) as the negative control. The DNA marker is a 100-bp or 1-kb ladder, as indicated. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (B) Schematic representation of primer positions for derivation of PCR products in FIV-infected cells derived from linear and circular viral DNA is shown. (C) One-LTR circle junction fragment homology (shaded segments) is indicated. As shown at the bottom of panel C, the fragments produced after FIV 34TF10 infection were colinear with that expected from the FIV 34TF10 genome.
FIG. 2
FIG. 2
Lack of FIV DNA in viral stocks and impact of AZT on the production of viral DNA intermediates. (A) Viral inocula were examined for the presence of viral DNA by PCR. Viral stocks corresponded to FIV-A grown in CrFK cells (AC), FIV-B grown in CrFK cells (BC), FIV-B grown in PBMC (BP), FIV-C grown in PBMC (CP), and FIV 34TF10 (34) grown in CrFK cells. FIV+ corresponds to a positive control (FIV-infected CrFK DNA). (B) CrFK cells were infected with FIV 34TF10 in replicate wells, differing only by the addition of AZT-1MP. Cells were harvested at 0, 24, 72, and 144 h and immediately lysed and stored for subsequent DNA extraction. PCR was performed using 50 ng of DNA as a template. PCRs were separated by agarose gel electrophoresis and visualized by ethidium bromide staining (10 μl per sample). The marker (MW) for the LTR and LTR-gag fragments was a 100-bp ladder and for the circle and β-actin fragments was a 1-kB ladder. Controls were H2O plus reagents (H2O), CrFK DNA (CrFK), FIV 34TF10-infected CRFK DNA (FIV+), and p34TF10.
FIG. 3
FIG. 3
AZT can block circle formation in CrFK and U87-T4-CXCR4 cells infected with CrFK-derived FIV-A or FIV-B. FIV subtype A or FIV subtype B was used to infect CrFK, U87-T4, U87-T4-CCR5, and U87-T4-CXCR4 cell lines in replicate wells differing only by the addition of AZT. Cells were harvested, and viral sequences were PCR amplified and visualized as described in the legend to Fig. 2. The figure depicts the LTR PCR (A), the LTR-Gag PCR (B), the circle fragment PCR (C), and the β-actin PCR (D). The PCR for each fragment was performed concurrently for all four cell lines. Four controls were included for each amplification and were run in lane 2 (PCR CNTL) of the four gels in each panel, with the first gel of each panel containing the H2O control, the second gel containing the CrFK control, the third gel containing the 34TF10 CrFK control, and the fourth gel containing the 34TF10 plasmid. AC, FIV-A grown in CrFK cells; BC, FIV-B grown in CrFK cells.
FIG. 3
FIG. 3
AZT can block circle formation in CrFK and U87-T4-CXCR4 cells infected with CrFK-derived FIV-A or FIV-B. FIV subtype A or FIV subtype B was used to infect CrFK, U87-T4, U87-T4-CCR5, and U87-T4-CXCR4 cell lines in replicate wells differing only by the addition of AZT. Cells were harvested, and viral sequences were PCR amplified and visualized as described in the legend to Fig. 2. The figure depicts the LTR PCR (A), the LTR-Gag PCR (B), the circle fragment PCR (C), and the β-actin PCR (D). The PCR for each fragment was performed concurrently for all four cell lines. Four controls were included for each amplification and were run in lane 2 (PCR CNTL) of the four gels in each panel, with the first gel of each panel containing the H2O control, the second gel containing the CrFK control, the third gel containing the 34TF10 CrFK control, and the fourth gel containing the 34TF10 plasmid. AC, FIV-A grown in CrFK cells; BC, FIV-B grown in CrFK cells.
FIG. 4
FIG. 4
FIV antigen production corresponds to circle formation during FIV infection. Lysed cells and supernatants were examined at days 10 and 17 PI. (A) Circle and β-actin fragment PCR results from days 10 and 17 PI. The positive control was 34TF10 CrFK DNA and the negative control was an H2O reagent control. The PCR controls were run in lane 2 (PCR CNTL), with the H2O reagent control on the top gel for each fragment and the positive control on the bottom gel for each fragment. The DNA marker was the 1-kb ladder. (B) Results from the enzyme-linked immunosorbent assays for FIV antigen. T4, U87-T4 cells; R5, U87-T4-CCR5 cells; X4, U87-T4-CXCR4 cells. Virus designations are defined in the legend to Fig. 2.
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