P. D'Aloja et al., Genetic and functional analysis of the human immunodeficiency virus (HIV) type 1-inhibiting F12-HiVnef allele, J GEN VIROL, 82, 2001, pp. 2735-2745
The primary human immunodeficiency virus type 1 (HIV-1) Nef mutant F12-HIVN
ef is characterized by three rare amino acid substitutions, G(140)E, (VL)-L
-153 and E(177)G. It was reported previously that the expression of F12-HIV
Nef in the context of the highly productive NL4-3 HIV-1 strain blocks virus
replication at the level of virus assembly and/or release by a mechanism d
epending on the presence of the CD4 intracytoplasmic tail. Here, it is repo
rted that NL4-3 HIV-1 strains expressing F12-HIVnef alleles that were back-
mutated in each amino acid substitution readily replicated in CD4(+) cells.
Attempting to correlate possible functional alterations with antiviral eff
ects, both F12-HIVNef and its back mutants were tested in terms of well-cha
racterized markers of Nef expression. Both F12-HIVNef and its G(177)E back
mutant did not down-regulate CD4 as the consequence of a greatly reduced ra
te of CD4 internalization. On the other hand, F12-HIVNef as well as the E(1
40)G and (LV)-V-153 back mutants failed to activate the p62 Nef-associated
kinase (p62NAK). Thus, only F12-HIVNef was defective in both accelerated ra
tes of CD4 internalization and p62NAK activation, whereas at least one Nef
function was restored in all of the back mutants. Infection of cells expres
sing Nef-resistant CD4 molecules with HIV-1 strains encoding F12-HIVNef bac
k mutants showed that both the lack of accelerated CD4 endocytosis and an,
as yet, still unidentified function are required for the F12-HIVNef inhibit
ory phenotype. These results provide a detailed functional analysis of the
F12-HIVnef allele and support the idea that both CD4 accelerated internaliz
ation and p62NAK activation are part of the essential steps in the virus re
plication cycle.