Virion-targeted viral inactivation of human immunodeficiency virus type 1 by using Vpr fusion proteins

Abstract

Inactivation of progeny virions with chimeric virion-associated proteins represents a novel therapeutic approach against human immunodeficiency virus (HIV) replication. The HIV type 1 (HIV-1) Vpr gene product, which is packaged into virions, is an attractive candidate for such a strategy. In this study, we developed Vpr-based fusion proteins that could be specifically targeted into mature HIV-1 virions to affect their structural organization and/or functional integrity. Two Vpr fusion proteins were constructed by fusing to the first 88 amino acids of HIV-1 Vpr the chloramphenicol acetyltransferase enzyme (VprCAT) or the last 18 C-terminal amino acids of the HIV-1 Vpu protein (VprIE). These Vpr fusion proteins were initially designed to quantify their efficiency of incorporation into HIV-1 virions when produced in cis from the provirus. Subsequently, CD4+ Jurkat T-cell lines constitutively expressing the VprCAT or the VprIE fusion protein were generated with retroviral vectors. Expression of the VprCAT or the VprIE fusion protein in CD4+ Jurkat T cells did not interfere with cellular viability or growth but conferred substantial resistance to HIV replication. The resistance to HIV replication was more pronounced in Jurkat-VprIE cells than in Jurkat-VprCAT cells. Moreover, the antiviral effect mediated by VprIE was dependent on an intact p6(gag) domain, indicating that the impairment of HIV-1 replication required the specific incorporation of Vpr fusion protein into virions. Gene expression, assembly, or release was not affected upon expression of these Vpr fusion proteins. Indeed, the VprIE and VprCAT fusion proteins were shown to affect the infectivity of progeny virus, since HIV virions containing the VprCAT or the VprIE fusion proteins were, respectively, 2 to 3 times and 10 to 30 times less infectious than the wild-type virus. Overall, this study demonstrated the successful transfer of resistance to HIV replication in tissue cultures by use of Vpr-based antiviral genes.

FIG. 1

Expression and incorporation of Vpr fusion proteins into HIV-1 virions. HxBRU, HxBRU-VprIE, and HxBRU-VprCAT were transfected into COS-7 cells. At 72 h later, cell lysates and pelleted virions were analyzed by radioimmunoprecipitation. Samples were probed with an HIV-1-positive human serum combined with either anti-Vpr, anti-Vpu, or anti-CAT antibodies as indicated.

FIG. 2

Characterization of Jurkat cells expressing Vpr fusion proteins. (A) Analysis of vprIE- and vprCAT-specific mRNA expression in transduced Jurkat cells by Northern blotting with a vpr-specific probe. (B) CAT activity associated with HIV-1 virions produced from Jurkat-puro or Jurkat-VprCAT cell lines. CAT assays were performed with 20% sucrose cushion-purified virus isolated from Jurkat-VprCAT cells infected with HxBRU (Vpr⁺) or HxBRU-R- (Vpr⁻). (C) Effect of Vpr fusion protein expression on cell division. 293T cells were transfected with pCEPVpr, pCEPVpr-R-, pCEPVprIE, or pCEPVprCAT expression vectors and cultured in the presence of G-418 for 10 days. The number of G-418-resistant cell clones was then established. Error bars indicate standard deviations of three independent experiments.

FIG. 3

HIV replication in transduced Jurkat cells. Jurkat-puro, Jurkat-VprIE, and Jurkat-VprCAT cell lines were challenged with HxBRU (Vpr⁺) and HxBRU-R- (Vpr⁻) (100,000 cpm of RT activity) to assess their susceptibility to viral infection. The infection kinetics were monitored by measuring viral production as RT activity in the supernatant over time for Jurkat-VprCAT cells (A), Jurkat-VprIE cells (B), Jurkat-VprIE cells upon infection with 300,000 cpm (C) or 25,000 cpm (D), and Jurkat-VprIE and Jurkat-RevM10 cells upon infection with 100,000 cpm (E). The experiments presented here were performed two or three times and were found to be highly reproducible.

FIG. 4

Replication of p6^gag HIV mutants in Jurkat-VprIE cells. Jurkat-VprIE or control Jurkat-puro cells were infected with 300,000 cpm of p6^gag mutant HxB89LF-PS-R- or with 300,000 cpm of wild-type p6 isogenic virus HxB89-R-. Viral production was monitored over time by measurement of RT activity in the supernatant. The experiments presented here were performed three times and were found to be highly reproducible.

FIG. 5

Evaluation of viral infectivity. The infectious potential of HIV virions containing Vpr fusion proteins was analyzed by a MAGI cell assay. Briefly, HeLa-CD4-LTR/β-Gal indicator cells were infected with equivalent amounts of virus produced from Jurkat cells or Jurkat-VprCAT and Jurkat-VprIE cells infected with HxBRU (Vpr⁺) and HxBRU-R- (Vpr⁻). At 24 h postinfection, the number of blue colonies was evaluated. The experiment was performed in triplicate with three different virus stocks, and the standard deviations are indicated.