HIV-1 Vif inhibits G to A hypermutations catalyzed by virus-encapsidated APOBEC3G to maintain HIV-1 infectivity

Abstract

Background: HIV-1 viral infectivity factor (Vif) is an essential accessory protein for HIV-1 replication. The predominant function of Vif is to counteract Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G, A3G), a potent host restriction factor that inhibits HIV-1 replication. Vif mediates the proteasomal degradation of A3G and inhibits A3G translation, thus diminishing the pool of A3G that is available to be packaged into budding virion. Although Vif is robust in degrading A3G, the protection provided against A3G is not absolute. Clinical and laboratory evidence have shown that A3G is not completely excluded from HIV-1 viral particles during HIV-1 replication. It remains unclear why the viral samples are still infectious when A3G has been packaged into the virions.

Results: In this study, we provide evidence that Vif continues to protect HIV-1 from the deleterious effects of A3G, even after packaging of A3G has occurred. When equal amounts of A3G were packaged into budding virions, the virus expressing functional Vif was more infectious and incurred fewer G to A hypermutations in the second round of infection compared to Vif-deficient virus. A Vif mutant with a defect in viral packaging showed a reduced ability to protect the HIV-1 genome from G to A hypermutations.

Conclusion: Our data suggest that even packaged A3G is still under the tyranny of Vif. Our work brings to light an additional caveat for any therapy that hopes to exploit the Vif-A3G axis. The ideal strategy would not only enhance A3G viral packaging, but also reduce HIV-1 Vif viral encapsidation.

Figure 1

A3G antiviral activity is circumvented more efficiently with HXB2N compared to HXB2NΔVif. 4 μg HXB2N or HXB2NΔVif proviral construct DNA was cotransfected with indicated amount of A3G expression vector DNA into 293T cells. 4 μg pcDNA3.1 was also cotransfected with HXB2N or HXB2NΔVif into 293T cells as controls. At 48 h after transfection, cell culture supernatants were harvested and used to infect TZM-bl indicator cells to measure viral infectivity (upper panel). The concentration of viral input was normalized by p24 ELISA. Virions in cell culture supernatants were precipitated by ultracentrifugation for Western blot analysis (lower panel). The percentage represents the quantity of intraviral A3G normalized by the quantity of intraviral p24. A3G/HXB2NΔVif was set as 100%. Each experiment in this and subsequent figures was performed at least three times, and representative results are presented.

Figure 2

HIV-1 Vif destroys A3G cytidine deaminase activity in both cells and virions. 293T cells were cotransfected with HAA3G22K or A3GD128K in combination with HXB2N, HXB2NΔVif or HXB2B3. At 48 h post-transfection, the cells were collected, and virions in culture supernatants were precipitated by ultracentrifugation for Western blot analysis (A). HAA3G22K was cotransfected into 293T cells with HXB2N, HXB2NΔVif or HXB2B3. At 48 h post-transfection, cells and culture supernatants were harvested to measure A3G cytidine deaminase activity (B). A3GD128K was cotransfected into 293T cells with HXB2N, HXB2NΔVif or HXB2B3. At 48 h post-transfection, cells and culture supernatants were harvested to measure A3G cytidine deaminase activity (C).

Figure 3

Vif counteracts the ability of A3G to induce G to A hypermutations. HXB2N, HXB2NΔVif or HXB2B3 was cotransfected with A3G, HAA3G22K or A3GD128K into 293T cells. At 48 h post-transfection, culture supernatants were harvested, and virus particles were precipitated by ultracentrifugation for Western blot analysis (A). The cell culture supernatants were also used to infect TZM-bl indicator cells to measure viral infectivity (B). The cell culture supernatants from panel A were used to infect SupT1 cells for hypermutation analysis (C).

Figure 4

VifB3 mutant encapsidation into HIV particles is defective. 293T cells were cotransfected with A3G or HAA3G22K and a proviral construct HXB2N, HXB2NΔVif or HXB2B3. At 48 h post-transfection, cells and culture supernatants were harvested, and viral particles were precipitated by ultracentrifugation for Western blot analysis (A). HXB2N, HXB2NΔVif or HXB2B3 was transfected into 293T cells. At 48 h post-transfection, cells were collected, and viral particles in culture supernatants were precipitated by ultracentrifugation for Western blot analysis (B). The percentage represents the quantity of intraviral Vif normalized by the quantity of intraviral p24. HXB2N was set to 100%.

Figure 5

HXB2N virus replicates more efficiently than HXB2NΔVif or HXB2B3 in Jurkat cell lines stably expressing HAA3G22K. Jurkat cells stably expressing A3G or HAA3G22K were analyzed by Western blot (A). Virus derived from HXB2N, HXB2NΔVif or HXB2B3 was used to infect Jurkat (B), Jurkat/A3G (C) and Jurkat/HAA3G22K (D) stable cell lines. Viral supernatants were harvested on days 1, 3, 5 and 7 post-infection and analyzed by p24 ELISA to measure HIV-1 levels.