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

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.

Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications

“…The results also demonstrate that the NC domain in Gag is crucial for its chaperone function in strand transfer, in accord with studies on primer placement with Gag (Cen et al, 2000;Feng et al, 1999;Guo et al, 2009) and Gag-derived proteins (Chan et al, 1999;Roldan et al, 2005). In vitro, mature NC also promotes primer placement (Cen et al, 2000;Feng et al, 1999;Hargittai et al, 2001;Iwatani et al, 2007;Prats et al, 1988;Rong et al, 1998;Tisné et al, 2001); for more detailed discussion and references, see Levin et al, 2005).…”

“…For example, the MA domain is responsible for Gag targeting to the plasma membrane during virus assembly and also binds RNA (Adamson and Freed, 2007;Chukkapalli et al, 2010;Shkriabai et al, 2006). CA is a protein interaction domain that facilitates Gag multimerization (Datta et al, 2007a,b;Gamble et al, 1997); the NC domain binds RNA, is required for genomic RNA packaging and primer placement (Darlix et al, 1995;Kleiman and Cen, 2004;Levin et al, 2005;Rein et al, 1998), has a role in viral RNA dimerization (Darlix et al, 1990;Feng et al, 1999;Liang et al, 1998), and interacts with the Bro 1 domain of the host protein Alix during virus assembly (Dussupt et al, 2009;Popov et al, 2009); and the p6 domain is needed for virus release and interaction with host factors in the ESCRT pathway (Adamson and Freed, 2007). During or shortly after budding of virions from the cell, the viral protease (PR) is activated and Gag is cleaved into the virus structural proteins.…”

“…This activity allows NC to catalyze nucleic acid conformational changes that result in the most thermodynamically stable structures (Tsuchihashi and Brown, 1994;reviewed in Levin et al, 2005;Rajkowitsch et al, 2007;Rein et al, 1998). The NC domain in Gag also has nucleic acid chaperone activity and as mentioned above, its role in viral DNA synthesis is to mediate primer placement, i.e., annealing of the primer to the viral RNA genome, a reaction that occurs during virus assembly (Cen et al, 1999(Cen et al, , 2000Feng et al, 1999;Huang et al, 1997;Kleiman and Cen, 2004).…”

Comparative nucleic acid chaperone properties of the nucleocapsid protein NCp7 and Tat protein of HIV-1

“…Positioning the replication primer tRNA onto genomic RNA During assembly, cellular tRNA Lys isoacceptors are selectively incorporated into virions (Jiang et al, 1993;Mak and Kleiman, 1997;Pavon-Eternod et al, 2010). The chaperone properties of the NC domain in Pr55gag are thought to facilitate the specific placement of the tRNA Lys,3 primer on the primer binding site (PBS) of the viral RNA (Cen et al, 1999;Feng et al, 1999;Cruceanu et al, 2006b;Guo et al, 2009;Wu et al, 2010). In vitro, NCp7 directs the annealing of the tRNA Lys,3 primer to the PBS (Li et al, 1996), by facilitating the strand exchange at the level of the tRNA acceptor stem and by unlocking in the presence of the complementary genomic RNA sequence, the highly stable interactions at the level of the T C loop (Chan and Musier-Forsyth, 1997;Tisné et al, 2003Tisné et al, , 2004Hargittai et al, 2004;Tisné, 2005;Barraud et al, 2007).…”

tRNALys3: The Primer tRNA for Reverse Transcription in HIV‐1

“…In the latter report, it was also shown that nucleocapsid protein mutations in Pr55 gag had a much greater effect on inhibiting tRNA Lys3 annealing than nucleocapsid protein mutations in Pr160 gag-pol , indicating an important role for Pr55 gag in tRNA Lys3 annealing. It has also been shown that Pr55 gag alone is sufficient for annealing tRNA Lys3 to genomic RNA in vitro (54), and that in vivo, processing of Pr55 gag and Pr160 gag-pol is not required for annealing to the PBS (55).…”