Biological activity of human immunodeficiency virus type 1 Vif requires membrane targeting by C-terminal basic domains

Abstract

Human immunodeficiency virus type 1 (HIV-1) encodes a Vif protein which is important for virus replication and infectivity. Vif is a cytoplasmic protein which exists in both membrane-associated and soluble forms. The membrane-associated form is an extrinsic membrane protein which is tightly associated with the cytoplasmic side of membranes. We have analyzed the mechanism of membrane targeting of Vif and its role in HIV-1 replication. Mutagenesis studies demonstrate that C-terminal basic domains are required for membrane association. Vif mutations which disrupt membrane association also inhibit HIV-1 replication, indicating that membrane localization of Vif is likely to be required for its biological activity in vivo. Membrane binding of Vif is almost completely abolished by trypsin treatment of membranes. These results demonstrate that membrane localization of Vif requires C-terminal basic domains and interaction with a membrane-associated protein(s). This interaction may serve to direct Vif to a specific cellular site, since immunofluorescence staining and plasma membrane fractionation studies show that Vif is localized predominantly to an internal cytoplasmic compartment rather than to the plasma membrane. The mechanism of membrane targeting of Vif is different in some respects from that of other extrinsic membrane proteins, such as Ras, Src, and MARCKS, which utilize a basic domain together with a lipid modification for membrane targeting. Membrane targeting of Vif is likely to play an important role in HIV-1 replication and thus may be a therapeutic target.

Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vif Protein

“…4 and 5). It has also been reported that the combined action of a number of positively charged amino acids toward Vif's carboxy terminus contributes to function (2,11,13). Although the substitution of the lysines at positions 157 and 160 had no effect on activity (Fig.…”

Characterization of Human Immunodeficiency Virus Type 1 vif Gene in Long-Term Asymptomatic Individuals

“…Between these regions, conserved motifs were observed, in particular the SLQXLA motif located between amino acids 144 and 149 (Oberste and Gonda, 1992) and two other motifs in the last 30 C-terminal amino acids of the Vif protein, i.e., 161 PPLPS 165 and 168 KLTEDRWN 175 . These two latter motifs are located in the domain which is in association with the plasma membrane and with the Gagprecursor (Bouyac et al, 1997;Goncalves et al, 1995). Moreover Arg and Lys residues within the C-terminal end of Vif (8 to 11 residues between the positions 157 and 192), which are the most important residues for those interactions, are largely conserved in all LTNP as well as in late progressor Vif sequences.…”

Cytoskeleton association and virion incorporation of the human immunodeficiency virus type 1 Vif protein

“…Nevertheless, it was suggested that Vif is required at the time of particle production in the host cell for regulating virus assembly or maturation (2,3,11,35,49). Vif has been shown to associate with the cytoplasmic face of cellular membranes (17), a phenomenon that involves basic residues located in the C-terminal portion of Vif (18). Vif muta-tions that disrupt membrane association also affect Vif function (18), suggesting a correlation between membrane binding and Vif activity.…”

“…Vif has been shown to associate with the cytoplasmic face of cellular membranes (17), a phenomenon that involves basic residues located in the C-terminal portion of Vif (18). Vif muta-tions that disrupt membrane association also affect Vif function (18), suggesting a correlation between membrane binding and Vif activity.…”

“…We therefore conclude that the partitioning of Vif with the cytoskeletal fraction is not due to nonspecific aggregation of Vif but is an inherent feature of this viral protein and most likely reflects the association of Vif with cytoskeletal components. It should be noted that previous studies investigating the subcellular distribution of Vif reported a significant amount of Vif associated with cellular membranes (17,18,32). However, these studies did not analyze cytoskeletal fractions of cells and thus ignored the portion of Vif that partitions with that fraction.…”