The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site - PubMed (original) (raw)
The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA and placement of tRNA on the primer binding site
Y X Feng et al. J Virol. 1999 May.
Abstract
The formation of an infectious retrovirus particle requires several RNA-RNA interaction events. In particular, the genomic RNA molecules form a dimeric structure, and a cellular tRNA molecule is annealed to an 18-base complementary region (the primer binding site, or PBS) on the genomic RNA, where it will serve as primer for reverse transcription. tRNAs normally possess a highly stable secondary and tertiary structure; it seems unlikely that annealing of a tRNA molecule to the PBS, which involves unwinding of this structure, could occur efficiently at physiological temperatures without the assistance of a cofactor. Many prior studies have shown that the viral nucleocapsid (NC) protein can act as a nucleic acid chaperone (i.e., facilitate annealing events between nucleic acids), and the assays used to demonstrate this activity include its ability to catalyze dimerization of transcripts representing retroviral genomes and the annealing of tRNA to the PBS in vitro. However, mature NC is not required for these events in vivo, since protease-deficient viral mutants, in which NC is not cleaved from the parental Gag polyprotein, are known to contain dimeric RNAs with tRNA annealed to the PBS. In the present experiments, we have tested recombinant human immunodeficiency virus type 1 Gag polyprotein for nucleic acid chaperone activity. The protein was positive by all of our assays, including the ability to stimulate dimerization and to anneal tRNA to the PBS in vitro. In quantitative experiments, its activity was approximately equivalent on a molar basis to that of NC. Based on these results, we suggest that the Gag polyprotein (presumably by its NC domain) catalyzes the annealing of tRNA to the PBS during (or before) retrovirus assembly in vivo.
Figures
FIG. 1
Demonstration of nucleic acid chaperone activity by selective annealing of oligonucleotides. (A) Oligonucleotides were incubated alone (lanes 1 and 2) or with 0.01, 0.04, or 0.16 μg of NC (lanes 3 and 4, 5 and 6, 7 and 8, respectively) or 0.02, 0.08, 0.32, or 1.3 μg of GagΔp6 (lanes 9 and 10, 11 and 12, 13 and 14, and 15 and 16, respectively) at 0° (odd-numbered lanes) or 37°C (even-numbered lanes) for 60 min. Lanes 17 to 19 represent markers, in which the labeled oligonucleotide was mixed with nothing (lane 17), with the 28-base complementary oligonucleotide alone (lane 18), or with the 21-base complementary oligonucleotide alone (lane 19), heated to 90°C for 5 min, and allowed to cool slowly to room temperature. (B) Data from incubations at 37°C in panel A as analyzed by phosphorimaging.
FIG. 2
Annealing of complementary RNAs in the presence of Gag or NC. RNAs were incubated with nothing (lane 12) or with 200 ng of BSA/μl (lanes 1 and 2), 330 ng of NC/μl (lanes 3 to 6), or 2.7 μg of Gag/μl (lanes 8 to 11) for 1 h (lanes 1, 3, 5, 8, and 10) or 4 h (lanes 2, 4, 6, 9, 11, and 12) and analyzed as described in Materials and Methods. RNA which was complementary to the labeled RNA was present in lanes 1 to 4 and 7 to 9. The samples were heated to 90°C for 5 min; those shown in lanes 7 and 12 (indicated by an asterisk) were allowed to cool slowly to room temperature, while the others were quickly chilled on ice before incubation as indicated. ss, single stranded; ds, double stranded.
FIG. 3
Dimerization of HIV 1-331 transcripts in the presence of Gag or NC. 32P-labeled HIV-1 1-331 RNA, prepared as described in Materials and Methods, was incubated with 0, 0.01, 0.02, 0.05, 0.10, 0.50, 1.0, 1.25, or 1.5 μg of NC (lanes 1 to 9) or 0, 0.08, 0.16, 0.4, 0.8, 4.0, 8.0, 10.0, or 12.0 μg of Gag (lanes 10 to 18). The results were analyzed by electrophoresis and autoradiography as described in Materials and Methods.
FIG. 4
Stabilization of dimers of HaSV 34-378 RNA by incubation with Gag or NC. A total of 0.5 μg of [32P]-labeled HaSV 34-378 RNA was incubated at 37°C with 2 μg of BSA (lanes 1 to 7), 1.5 μg of NC (lanes 8 to 14), or 12 μg of GagΔp6 (lanes 15 to 21). Thermostabilities of the dimers were then determined by incubation for 10 min at 0° (lanes 1, 8, and 15), 25° (lanes 2, 9, and 16), 37° (lanes 3, 10, and 17), 45° (lanes 4, 11, and 18), 55° (lanes 5, 12, and 19), 65° (lanes 6, 13, and 20), or 70° (lanes 7, 14, and 21), followed by electrophoresis and autoradiography as described in Materials and Methods. Some RNA remained at the top of the gel in lanes 15 to 17, but this aggregation was not a consistent feature of these experiments.
FIG. 5
Placement of tRNA3Lys on HIV-1 1-331 RNA by GagΔp6. (A) 32P-labeled tRNA3Lys was incubated with retroviral transcript and GagΔp6, and the reactions were analyzed as described in Materials and Methods. Lanes: 1, tRNA alone; 2, tRNA plus HaSV 34-378 plus 6 μg of GagΔp6; 3, tRNA plus HIV-1 1-331 plus 3 μg of GagΔp6; 4, tRNA plus HIV-1 1-331 plus 6 μg of GagΔp6; 5, tRNA plus HIV-1 1-331. (B) Unlabeled tRNA3Lys was annealed to the PBS on HIV-1 1-331 RNA by incubation as indicated. The mixtures were deproteinized as described in Materials and Methods and then tested for the presence of primer on the HIV-1 template RNA by addition of reverse transcriptase and dNTPs, including [α-32P]dCTP. The reactions were analyzed as described in Materials and Methods. Size of the labeled DNA product was determined ± 10 nt compared with labeled DNAs of known sizes (data not shown). Lanes: 1, tRNA plus 6 μg of GagΔp6; 2, 6 μg of GagΔp6 plus HIV-1 1-331; 3, tRNA plus HIV-1 1-331; 4, tRNA plus 6 μg of GagΔp6 plus HIV-1 1-331.
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