Cell Cycle Arrest by Vpr in HIV-1 Virions and Insensitivity to Antiretroviral Agents
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; X. F. Yu M. Matsuda M. Essex T. H. Lee ibid. p. 5688; X. Yuan
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; J. Hoch et al. ibid. p. 4807.
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J. B. M. Jowett et al. ibid. p. 6304.
Poon B., et al., ibid. 71, 3961 (1997).
S. Stewart B. Poon J. B. M. Jowett I. S. Y. Chen ibid. p. 5579.
B. Poon and I. S. Y. Chen unpublished observations.
All virus stocks were generated by calcium phosphate-mediated transfection [J. Sambrook E. F. Fritsch T. Maniatis in Molecular Cloning N. Ford C. Nolan M. Ferguson Eds. (Cold Spring Harbor Laboratory Press Cold Spring Harbor NY ed. 2 1989) pp.16.32–16.34] of 293T cells. VSV-G pseudotyped HIV-1 NL4-3-thy env(−)vprX and HIV-1 NL4-3-thy env(−) were generated after transfection with pCMV-VSV-G and either NLthyΔBglVprX or NLthyΔBgl and then concentrated ( 9 ). Virus stocks of HIV-1 NL4-3-thy env(−)vprX + BSVpr were generated after transfection with BSVpr [
] pCMV-VSV-G and NLthyΔBglVprX.
Virus defective for de novo expression of all HIV-1 genes but that packages Vpr was obtained with pHR'CMV-thy in which the lacZ gene from pHR'-CMV-lacZ [
] was replaced by the Thy 1.2 gene pCMVΔR8.2 [
Naldini L., Blomer U., Gage F. H., Trono D., Verma I. M., Proc. Natl. Acad. Sci. U.S.A. 93, 11383 (1996);
] and pCMV-VSV-G and concentrated ( 9 ). PBLs isolated from normal uninfected donors were stimulated for 72 hours in RPMI 1640 with 20% fetal calf serum (FCS) and phytohemagglutinin (5 mg/ml) and were maintained after infection in RPMI 1640 with 10% FCS and recombinant interleukin-2 (20 units/ml). At 72 hours after infection infection of PBLs with virus containing Vpr resulted in a decrease in the G 1 /G 2 ratio of Thy 1.2 + cells compared with mock-infected cells (1.70 and 6.58 respectively). At 72 hours after infection infection of SupT1 T cells resulted in a decrease in the G 1 /G 2 ratio of Thy 1.2 + cells compared with mock-infected cells (0.43 and 1.7 respectively).
Virions pseudotyped with HIV-1 LAI envelope were obtained after transfection with pHR'CMV-thy pCMVΔR8.2 and pLET-LAI [
] and concentrated by ultracentrifugation at 40 000 g for 1 hour. At 72 hours after infection of HeLa CD4 cells the levels of infection were low (3.3% Thy 1.2 + cells) but in Thy 1.2 + cells infection with virus containing Vpr resulted in a decrease in the G 1 /G 2 ratio compared with mock-infected cells (1.4 and 2.4 respectively).
Nonenveloped viral particles were obtained after transfection with NLthyΔBgl and concentrated ( 9 ). Vpr was detected at comparable levels in the enveloped and nonenveloped viral preparations by Western blot analysis ( 28 ). At 48 hours after infection HeLa cells infected with nonenveloped HIV-1 had a cell cycle profile comparable to that of mock-infected cells (G 1 /G 2 ratios of 2.1 and 2.25 respectively). Infection with VSV-G–enveloped HIV-1 in parallel resulted in a G 1 /G 2 ratio of 0.15.
Infection of HeLa cells with HIV-1 that packages Vpr ( 13 ) sufficient to productively infect 12% of the cells (12% Thy 1.2 + ) resulted in a decrease in the G 1 /G 2 ratio of Thy 1.2 + cells compared with mock-infected cells (0.61 and 2.1 respectively). Similarly infection of SupT1 T cells to obtain 6% Thy 1.2 + cells resulted in a decrease in the G 1 /G 2 ratio of Thy 1.2 + and Thy 1.2 – cells compared with mock-infected cells (0.66 and 0.76 and 1.4 respectively).
For experiments involving RTI cells were treated with 5 μM 3′-azido-3′-deoxythymidine (Sigma) and 5 μM nevirapine for 2 hours before infection and RTIs were maintained in the medium during and after infection. RTI treatment of mock-infected or VSV-G or HIV-1 LAI pseudotyped vprX mutant-infected cells resulted in G 1 /G 2 ratios of 1.64 1.27 and 1.35 respectively compared with 1.75 for non-RTI–treated cells. Virions pseudotyped with HIV-1 envelope were obtained after transfection with NLthyΔBgl or NLthyΔBglVprX and pLET-LAI. Supernatant was collected 48 hours after transfection and concentrated by ultracentrifugation at 40 000 g for 1 hour.
Virus produced in the presence of PI was obtained after transfection with either NLthyΔBglVprX or NLthyΔBgl and pCMV-VSV-G. At 24 hours after transfection the medium was replaced with medium containing 100 nM indinavir sulfate (Merck). Supernatant was collected 48 and 72 hours after transfection and concentrated ( 9 ).
K. Tenner-Racz et al. in Progress in AIDS Pathology H. Rotterdam and S. C. Sommers Eds . (Field and Wood New York 1989) pp. 29–40;
; G. Pantaleo et al. ibid. p. 355.
; D. Finzi et al. ibid. p. 1295; J. K. Wong et al. Proc. Natl. Acad. Sci. U.S.A. 94 12574 (1997) T.-W. Chun et al. ibid. p. 13193.
Concentrated virus was lysed in 2× loading buffer and subjected to electrophoresis on SDS–15% polyacrylamide gels ( 9 ). Western blotting was performed with a rabbit polyclonal antibody for Vpr (provided by N. Landau Aaron Diamond AIDS Research Center New York) or human anti-HIV hyperimmune plasma (provided by P. Krogstad and Y. Bryson UCLA) and developed with the enhanced chemiluminescence assay (Amersham Arlington Heights IL).
The method of double staining for surface marker Thy 1.2 and DNA content was performed as described in ( 9 10 ). All stained cells were acquired on a FACScan II apparatus (Becton-Dickinson) and analyzed with Cell Quest software.
We thank D. S. An J. Zack and P. Krogstad for valuable reagents and advice and Vaheideh Gudeman for technical support. Supported by NIH grant CA70018 and Center for AIDS Research grant AI28697. B.P. and K.G.-F. were supported by NIH postdoctoral training grant T32/A107388.