The incorporation of tRNALys
isoacceptors into HIV-1 requires Gag-Pol (36
). C-terminal deletions which include sequences coding for integrase and the RNase H and connection domains of RT do not inhibit tRNALys
packaging, but further deletions into the thumb domain of RT inhibit tRNALys
selective packaging (31
). We have investigated whether Pol sequences alone can facilitate the selective incorporation of tRNALys
isoacceptors into the virus. Cells were transfected with BH10.P− alone or BH10.FS− alone or were cotransfected with BH10.FS− and either hGag-PolΔFSΔPR or hPol. Western blot analysis similar to that shown in Fig. was used to determine the Gag-Pol or Pol/Gag ratios in the VLPs produced from these transfected cells, and these ratios are listed in Table . As previously demonstrated by the results shown in Fig. , Pol incorporation into Gag VLPs is somewhat lower (70 to 80%) than Gag-Pol incorporation.
The effect of Gag-Pol or Pol incorporation upon selective packaging and annealing of tRNA3Lys in HIV-1a
Total RNA was then isolated from virions, and analyzed by dot blot hybridization with probes specific for tRNA3Lys
or viral genomic RNA, as previously described (14
). These results are shown in Fig. , which also includes standard curves of different concentrations of genomic RNA or tRNA3Lys
to show that the dot blot signals of samples fall within the linear range of the curves. The ratios of tRNA3Lys
/genomic RNA were then determined, and these ratios, normalized to BH10P−, are also listed in Table . It can be seen that the lower amount of tRNA3Lys
packaged reflects the lower amount of Pol incorporated into VLPs, indicating that Pol sequences can efficiently replace Gag-Pol in facilitating tRNA3Lys
packaging into VLPs.
FIG. 3. Incorporation of tRNA3Lys into Gag VLPs and its annealing to viral RNA. HEK-293T cells were transfected or cotransfected with different plasmids coding for wild-type Gag and wild-type or mutant Gag-Pol proteins with Lipofectamine. The plasmids used are (more ...)
The ability of Pol sequences to selectively package tRNALys
isoacceptors into Gag VLPs was further analyzed by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), as previously described (28
). Total viral RNA was 3′-end-labeled with [32
P]pCp and analyzed by 2D-PAGE. Only low-molecular-weight RNA can enter the gel, and Fig. shows the 2D-PAGE patterns of this RNA. Panel I shows the pattern of RNA in virions produced from cells transfected with the BH10.P− plasmid. The labeled spots have previously been identified (28
); spot 3 is tRNA3Lys
, the primer tRNA for RT, and spots 1 and 2 represent tRNA1,2Lys
. A similar pattern of low-molecular-weight RNA is seen in VLPs produced from cells cotransfected with BH10.FS−, and either hGag-PolΔFSΔPR (Fig. , panel II), or hPol (panel III). In virions produced from cells transfected only with BH10.FS−, no selective packaging of tRNALys
isoacceptors is seen (panel IV).
The ability of Pol to selectively package primer tRNA3Lys
is also reflected in the ability of the total viral RNA to support reverse transcription. We have shown that the annealing of tRNA3Lys
to viral genomic RNA is proportional to the viral concentration of tRNA3Lys
). To measure the amount of tRNA3Lys
annealed to the primer binding site in vivo, total viral RNA is used as the source of primer or template in an in vitro reverse transcription reaction with exogenous HIV-1 RT, dCTP, dTTP, α-32
P-dGTP, and ddATP. This assay measures the amount of extendable tRNA3Lys
placed on the viral genome (it is not known if all annealed tRNA3Lys
is extendable). Since the sequence of the first six dNTPs incorporated is CTGCTA, annealed primer tRNA3Lys
will be extended by only six bases in the presence of ddATP, and the extended tRNA3Lys
can be resolved and detected by one-dimensional PAGE. The electrophoretic results are shown in Fig. and listed in Table . The first four lanes in Fig. use increasing amounts of genomic RNA in the viral RNA to show that the signal undergoes a linear increase with increasing RNA. The amount of viral genomic RNA labeled 1.00 in lane 4 is the amount of genomic RNA used in the remaining five lanes. The ratios of extension of primer tRNA3Lys
/genomic RNA, relative to that found for BH10P−, are listed in Table . tRNA3Lys
placement in Gag VLPs containing Pol is significantly reduced compared to VLPs containing Gag-Pol. In part this result is probably due to the reduced incorporation of tRNA3Lys
, since we have shown that the amount of tRNA3Lys
annealed to viral genomic RNA is proportional to the amount of tRNA3Lys
). However, the reduction in tRNA3Lys
annealing is greater than expected, which may reflect factors other than the concentration of tRNA3Lys
in the virion, including the presence of Pol rather than Gag-Pol in the virion.
In this report, we have shown that Pol can be efficiently incorporated into Gag VLPs independently of upstream Gag sequences in Gag-Pol. Although this process has been studied in a cellular system in which larger amounts of viral proteins are being synthesized compared to normally infected T cells, it is unlikely that Pol incorporation is the result of random packaging. Thus, the results of the experiment shown in Fig. demonstrate that Rous sarcoma Gag VLPs do not package HIV Pol species. Also, the results of the experiments shown in Fig. demonstrate a specific requirement for particular Gag sequences. Replacement of NC in Gag with a leucine zipper motif (plasmid Zwtp6) prevents Gag-Pol, but not Pol, incorporation into Gag VLPs, while an additional deletion of p6 in Gag (plasmid Zwt) reduces Pol incorporation into Gag VLPs four- to fivefold.
It is possible, however, that with lower viral protein concentrations in the cell, the interaction of Gag-Pol with Gag p6 plays a minor role in the incorporation of Gag-Pol into virions, with homologous interactions between Gag and Gag (Gag-Pol) being sufficient for this process. The interaction between Gag-Pol and Gag p6 may be more important at a later stage of assembly. Thus, previous reports have presented data that suggest that the HIV-1 Gag p6 domain functions to retain the Pol domain proteins within the assembling virus after the initial activation of the viral PR (11
). Current evidence suggests that prior to viral budding, the viral PR-induced cleavage of Gag-Pol at the cell membrane is initiated in trans
within a Gag-Pol dimer to produce an N-terminal Gag fragment terminating in p2 and a C-terminal fragment containing NC-transframe-PR-RT-integrase (42
). Thus, this first PR cleavage will separate that part of Gag sequences within Gag-Pol which contain major sites of binding for Gag, i.e., the major homology region and C-terminal portion of capsid. The binding of Pol sequences to p6 sequences in Gag could therefore be important for retaining Pol sequences and tRNALys
until viral budding is complete. This interpretation is further supported by the observation reported above that Pol can facilitate the selective packaging of tRNALys
isoacceptors with an efficiency reflecting the incorporation of Pol into the Gag particles (approximately 70% of that found using intact Gag-Pol) (Table ).