MLV glycosylated-Gag is an infectivity factor that rescues Nef-deficient HIV-1

Abstract

Optimal infectivity of HIV-1 virions requires synthesis of the HIV-1 regulatory protein Nef in some producer cells but not others. A survey of 18 lymphoid cell lines found that Nef was dispensable in three, each of which harbored gammaretroviruses. Nef-dependent cell lines were rendered Nef-independent by a cell-free supernatant from the independent lines or by transfection of cloned murine leukemia virus (MLV). Analysis of MLV deletion mutations identified glycosylated gag (glycogag) as the factor that rescues Nef-defective HIV-1 virions. Glycogag was also demonstrated to be required for the infectivity of MLV virions produced in lymphoid cells. Direct comparison of Nef and glycogag revealed identical dependence for activity on Env-pseudotype and producer cell type. The two proteins colocalize within cells, and both increase the yield of viral cDNA in target cells. The functional similarity of Nef and glycogag is a compelling example of convergent evolution in which two structurally unrelated proteins provide a function necessary for virion infectivity in lymphoid cells.

Fig. 1.

HIV-1 infectivity is Nef-independent when virions are produced from 3 of 18 lymphoid cell lines. (A) Fold enhancement of HIV-1_NL4-3 infectivity when virions are produced by using different cell lines. The dotted line denotes no enhancement. Data representative of two independent experiments. (B) The Nef activity on HIV-1 infectivity is redundant in “Nef-independent” producer cell lines. Infectivity of HIV-1 produced in the indicated cells lines was determined on TZM-bl indicator cells. Bars represent the means plus SD from triplicate determinations. Results are representative of two independent experiments.

Fig. 2.

MLV rescues the infectivity of HIV-1^Nef-. (A) Cell-free supernatant from JJ6 renders HIV-1 production from JTAg (JTAg/JJ6) Nef-independent. (B) MLV provirus in producer cells (JTAg+MLV) abolishes the Nef requirement for optimal HIV-1 infectivity. (B Upper) TZM-bl cells infected with RT-normalized HIV-1^wt and HIV-1^Nef- and stained with X-Gal. (B Lower) Quantification of HIV-1 infectivity. (C) MLV is only minimally active on the infectivity of HIV-1(VSV-G) pseudotypes. Infectivity is expressed as a percentage of the HIV-1^wt control and is the mean plus SD from triplicate determinations. Results are representative of two independent experiments.

Fig. 3.

MLV Glycogag rescues the infectivity of HIV-1^Nef-. (A) Mapping the determinant in MLV that substitutes for Nef. Deletions (Δ) and frameshifts (fs) in the MLV provirus (Left) and the effect on HIV-1 infectivity (Right). (B Left) A mutation that abolishes translation of gPr80 (MLV^gg-) impairs the ability of MLV to rescue HIV-1^Nef-. (B Right) Western blot of JTAg producer cells. (C) The gag sequence of gPr80 is not required for the effect on HIV-1 infectivity. Infectivity is expressed as a percentage of the HIV-1^wt. Bars are means plus SD from triplicate determinations. Results are representative of two independent experiments.

Fig. 4.

gPr80 is an MLV infectivity factor. (A) gPr80 promotes the infectivity of MLV-X and MLV-A produced from JTAg cells. (B) The requirement of gPr80 for infectivity of MLV-E depends on the target cell type. (C) Nef expressed in trans does not rescue the infectivity of MLV^gg- (Left) but rescues HIV-1^Nef- (Right). Infectivity is expressed as percentage of WT. Bars are the mean plus SD from triplicate determinations. Results are representative of four (A) and three (B and C) independent experiments.

Fig. 5.

The infectivity factors gPr80 and Nef exhibit similar phenotypes. The effect of Env pseudotype (A) and producer cell line (B) on the activity of gPr80 (Left) or Nef (Right). (C) The effect of gPr80 or Nef on steady-state levels of MLV and HIV-1 full-length viral cDNA at the indicated times after infection. Bars represent the mean plus SD from triplicate determinations. Infectivity is expressed as percentage of the WT. Results are representative of three (A) and two (B) independent experiments.