Despite significant progress in the characterization of Vif-induced defects of HIV virions, the molecular mechanism of Vif-regulated viral infectivity remains unclear. One of the critical yet unresolved issues is from what cellular—or viral—compartment Vif exerts its activity. Even though previous studies have clearly identified Vif within HIV virions (10
), its functional relevance has been questioned. Unlike Vpr, which is packaged into HIV particles through an interaction with the p6 component of Gag (12
), packaging of Vif was thought to be nonspecific (10
). In addition, the relatively low abundance of Vif in virions, which in some reports approached the limit of detection (70
), and the notion that levels of Vif packaging can vary depending on the intracellular expression level without affecting viral infectivity have led to the suggestion that virion incorporation of Vif may not be necessary for Vif function (56
Our data clearly demonstrate the presence of significant amounts of Vif in viruses irrespective of whether the viruses were derived from permissive HeLa cells or restrictive H9 cells. Packaging of Vif is, in fact, specific and is sensitive to mutations in Vif and dependent on the viral nucleocapsid protein as well as viral genomic RNA. Several lines of evidence support this conclusion. First, mutations in the nucleocapsid zinc finger domain reduce Vif packaging to background levels (Fig. ). Second, removal of an RNA packaging signal on the viral genomic RNA abolished packaging of Vif (Fig. ). Third, detergent extraction of HIV virions demonstrates that, in contrast to the viral envelope (gp41), matrix (MA), and capsid (CA) components, which are highly sensitive to detergent extraction, Vif and integrase were insensitive to detergent treatment (Fig. ). Interestingly, while both reverse transcriptase and nucleocapsid proteins were found to be partially sensitive to detergent extraction (Fig. and data not shown), the active reverse transcription complex capable of directing the synthesis of (−)ssDNA was completely resistant to detergent extraction. Despite the fact that more than 70% of NC and reverse transcriptase were removed by detergent treatment, synthesis of (−)ssDNA in our in vitro assay was equally efficient in untreated and detergent-treated virus preparations, indicating that the integrity of viral reverse transcription complexes were not affected by detergent extraction of viral components. This suggests that the reverse transcriptase and NC molecules released by detergent treatment either constitute excessive amounts of these proteins in virions not tightly associated with reverse transcription complexes or reflect the release of these proteins from defective viral particles.
Viral genomic RNA was also found to be resistant to detergent extraction. This is evidenced by the absence in the soluble fractions of detergent-treated virions of a 354-base reverse transcriptase product directed by the internal Z85 primer (Fig. A, compare lanes 1 and 2 with lane 3). While our data are consistent with a previous report demonstrating the impact of deletions in the NC zinc finger domain on packaging of Vif into virions in a recombinant baculovirus system (33
), our observation that Vif is more resistant to detergent extraction than NC or reverse transcriptase as well as the absence of Vif in C-Help virus preparations, is more consistent with an association of Vif with viral genomic RNA rather than (or in addition to) NC. Our data therefore clearly show that Vif is not a soluble component of virions, is not attached to the outside of virions, and is not attached to the viral envelope. Packaging of Vif through a nonspecific mechanism such as passive diffusion is thus unlikely.
In agreement with a previous study (56
), we found that the absolute amounts of Vif packaged into virions were affected by the intracellular expression levels (not shown). However, the relative amounts of virion-associated Vif appeared to be rather constant and amounted to about 12.5% of total Vif. This expression level-dependent export of Vif might explain the differences reported in the literature for the number of Vif molecules packaged per virion (10
). While point mutations in various regions of Vif were not found to affect its packaging (10
), we observed that larger deletions near the N terminus and in the center of the protein had a severe impact on Vif packaging. Deletion of an N-terminal segment in VifΔD (residues 23 to 43) doubled its packaging efficiency to 23% (Fig. ). This deletion was found to increase the intracellular solubility of Vif, presumably by reducing its reported interaction with vimentin (34
; unpublished observations) and could explain the increased packaging efficiency of VifΔD. In contrast, deletion of residues 75 to 114 in VifΔG almost completely blocked packaging of the mutant protein (Fig. ). These results are consistent with a previous report that identified a requirement for residues 68 to 81 in Vif for packaging into virus-like particles in a baculovirus system (33
). Interestingly, deletion of the same region in Vif was also shown to eliminate its RNA-binding activity, which is supported by our observation that the subcellular distribution of wild-type Vif but not VifΔG is RNase sensitive (Fig. C). These data further support the notion that packaging of Vif into virions involves an interaction with viral genomic RNA.
While our attempts to identify differences in the protein composition of wild-type and Vif-defective viruses revealed subtle, producer cell-dependent variations (Fig. ), we failed to observe Vif-dependent variations in the viral protein composition. These results are consistent with observations by other groups (15
). Also, our pulse-chase analysis in infected H9 cells did not reveal tangible differences in protein synthesis, processing, or release of viral proteins that could explain the reported effects of Vif on the structure or stability of viral cores (32
). In addition, previous studies did not find any effects of Vif on the levels of genomic RNA and tRNALys
) or on genomic RNA dimerization or stability of the RNA dimer linkage (25
). Thus, while it is conceivable that viral infectivity requires subtle posttranslational modifications of viral components by Vif, which could be catalyzed by intracellular Vif either before or during virus assembly, there is currently no experimental evidence to support such a mechanism. The obvious correlation between the packaging of viral genomic RNA and Vif and the specific association of Vif with viral cores make it tempting to speculate that packaging of Vif is functionally significant and required for infectivity of virions produced in restrictive producer cells. Our observation that approximately 12% of intracellular Vif molecules are packaged into progeny virions suggest that the packaging of Vif occurs with an efficiency very similar to that reported for HIV-1 Env, where only 5 to 15% of the Env precursor gp160 molecules were found to be transported to the cell surface for virion incorporation (67
Several possible mechanisms can be envisioned to explain how virus-associated Vif could regulate viral infectivity. First, it is conceivable that due to its affinity to viral RNA and Gag, Vif has a critical role in stabilizing viral nucleoprotein complexes. The function of Vif would therefore be to facilitate proper assembly and/or maturation of components of the viral cores. Accordingly, the absence of Vif would result in unstable, defective cores with reduced ability for efficient cDNA synthesis. Such a mechanism would be consistent with the observation that Vif-defective particles exhibit reduced stability of their nucleoprotein or reverse transcription complexes (18
) and are impaired in the reverse transcription of their genomes (13
). Alternatively, it is possible that Vif, due to its ability to associate with viral nucleoprotein complexes as well as the cytoskeleton (31
), functions as an adapter to link the viral nucleoprotein or preintegration complex to a cellular transport pathway to facilitate its transport to the nuclear membrane. Such nuclear targeting mechanisms have been reported for other viruses, including herpes simplex virus 1 (59
), human foamy virus (50
), and adenovirus (64
). For these viruses, incoming capsids are targeted to the nucleus in a microtubule-dependent mechanism. Interestingly, similar microtubule-dependent transport was recently observed for HIV-1 cores using green fluorescent protein-tagged HIV particles (T. Hope, personal communication). In addition, we have found that cells undergo a rapid change in their cytoskeletal organization immediately following infection by HIV-1, and we observed that the effect of Vif on the structure of vimentin is microtubule dependent (K. Strebel, unpublished observations). It is therefore possible that HIV, like other viruses, employs an active transport mechanism for nuclear targeting of its nucleoprotein complex. Although it is currently unclear if and how Vif would be involved in these events, it is conceivable that Vif functions to connect the viral core to a cytoskeleton-dependent cellular transport mechanism. Both models, i.e., the possible function of Vif in stabilizing viral cores and its proposed function in nuclear targeting of viral cores, would require only small amounts of Vif molecules but would depend on the presence of Vif in virions.