Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vif Protein

Abstract

Lentivirus Vif proteins are potent regulators of virus infectivity. However, relatively little is known about the functional domains, peptide motifs, or residues of any Vif protein. In this report, we present the first extensive mutagenesis analysis of the 192-amino-acid human immunodeficiency virus type 1 (HIV-1) Vif protein. A large number of scanning missense (mostly alanine substitution) and deletion mutations were introduced into the HIV-1HXB3 vif gene, and the resulting proteins were evaluated for the induction of virus infectivity as well as subcellular localization. The results show that amino acids dispersed throughout Vif’s linear sequence are important for function. However, because many of the inactive proteins also appear to be mislocalized, we suggest that many of them may actually be misfolded rather lacking an intracellular targeting signal. Interestingly, disruptions within an internal region spanning residues 114 to 146 give rise to mutant proteins that either retain function or are inactive but are not substantially mislocalized. We therefore speculate that this region, which harbors two essential cysteine residues and one essential serine residue, may contain aspects of a putative Vif effector domain.

Insight into the HIV-1 Vif SOCS-box–ElonginBC interaction

“…The SOCS-box contains a conserved SLQYLA motif (residues 144-149), called the BC-box, which interacts with EloBC. Mutation of this motif leads to the inactivation of Vif, indicating an essential role for this domain [14,22,36,39,40]. The SOCS-box also has a critical proline-rich motif (161PPLPS165, PPLPS motif) [41,42] downstream of the BC-box, whose molecular mechanism of action remains unclear.…”

The inhibition of assembly of HIV-1 virus-like particles by 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB) is counteracted by Vif and requires its Zinc-binding domain

“…To further analyze the role of the ZBD structure in the Vif anti-DSB activity, we constructed another mutant of recombinant Vif protein. Cysteine at position 133 in Vif is a residue essential for virus infectivity [ 62 , 63 ], for Zn coordinate formation and ZBD-associated functions in Vif [ 27 , 28 ]. We therefore generated mutation C133S in recombinant Vif, and tested mutant VifC133S in co-expression with Pr55Gag in control or DSB-treated Sf9 cells, as above.…”

Fully functional, naturally occurring and C-terminally truncated variant human immunodeficiency virus (HIV) Vif does not bind to HIV Gag but influences intermediate filament structure

“…Fractionation analyses of transfected cells in various detergents have indicated that the Vif protein can be differentially solubilized. Thus, some cellular Vif is soluble in aqueous buffers, some can be solubilized in weak detergents and some can only be dissolved in strong ionic detergent solutions (Goncalves et al, 1994(Goncalves et al, , 1995Karczewski & Strebel, 1996 ;Simon et al, 1999). It is not clear whether these solubility properties reflect specific subcellular localizations of expressed Vif.…”

“…It is not clear whether these solubility properties reflect specific subcellular localizations of expressed Vif. Thus, it has been controversially discussed that Vif may be associated with cellular membranes (Goncalves et al, 1994(Goncalves et al, , 1995, cytoskeletal components (Karczewski & Strebel, 1996) or insoluble components distinct from cytoskeletal components (Simon et al, 1999).…”

“…In this context, it is of note that in a subpopulation of transfected HeLa cells expressing Vif from a subvirus vector, the distribution of the intermediate filament protein, vimentin, was altered such that vimentin was present in perinuclear aggregates to which Vif also localized (Karczewski & Strebel, 1996). Concerning putative interactions with viral proteins, it has been reported that Vif colocalizes with Gag in infected cells (Simon et al, 1997(Simon et al, , 1999, whereby more recent evidence suggests that this may reflect independent targetting to the same subcellular compartment without a specific interaction (Simon et al, 1999). Furthermore, it has been reported that Vif specifically and strongly interacts with Gag in vitro (Bouyac et al, 1997).…”