Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Webb, Joseph A.; Jones, Christopher P.; Parent, Leslie J.; Rouzina, Ioulia; Musier‐Forsyth, Karin

doi:10.1261/rna.038869.113

Cited by 84 publications

(166 citation statements)

References 79 publications

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“…This finding differs from those for HIV-1, for which reports have shown that NC is salt sensitive (51-53), but Gag-facilitated annealing is much less sensitive to salt in this range (20,54). Experiments demonstrating that aggregation of NA by RSV NCp12 is abrogated by increasing concentrations of NaCl are consistent with our observation that the NA chaperone activity of NCp12 was salt dependent (55).…”

Section: Discussionsupporting

confidence: 65%

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Rye-McCurdy

Nadaraia-Hoke

Gudleski-O'Regan

et al. 2014

J Virol

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Host cell tRNAs are recruited for use as primers to initiate reverse transcription in retroviruses. Human immunodeficiency virus type 1 (HIV-1) uses tRNALys3 as the replication primer, whereas Rous sarcoma virus (RSV) uses tRNA Trp . The nucleic acid (NA) chaperone function of the nucleocapsid (NC) domain of HIV-1 Gag is responsible for annealing tRNA Lys3 to the genomic RNA (gRNA) primer binding site (PBS). Compared to HIV-1, little is known about the chaperone activity of RSV Gag. In this work, using purified RSV Gag containing an N-terminal His tag and a deletion of the majority of the protease domain (H6.Gag.3h), gel shift assays were used to monitor the annealing of tRNA Trp to a PBS-containing RSV RNA. Here, we show that similar to HIV-1 Gag lacking the p6 domain (Gag⌬p6), RSV H6.Gag.3h is a more efficient chaperone on a molar basis than NC; however, in contrast to the HIV-1 system, both RSV H6.Gag.3h and NC have comparable annealing rates at protein saturation. The NC domain of RSV H6.Gag.3h is required for annealing, whereas deletion of the matrix (MA) domain, which stimulates the rate of HIV-1 Gag⌬p6 annealing, has little effect on RSV H6.Gag.3h chaperone function. Competition assays confirmed that RSV MA binds inositol phosphates (IPs), but in contrast to HIV-1 Gag⌬p6, IPs do not stimulate RSV H6.Gag.3h chaperone activity unless the MA domain is replaced with HIV-1 MA. We conclude that differences in the MA domains are primarily responsible for mechanistic differences in RSV and HIV-1 Gag NA chaperone function. IMPORTANCEMounting evidence suggests that the Gag polyprotein is responsible for annealing primer tRNAs to the PBS to initiate reverse transcription in retroviruses, but only HIV-1 Gag chaperone activity has been demonstrated in vitro. Understanding RSV Gag's NA chaperone function will allow us to determine whether there is a common mechanism among retroviruses. This report shows for the first time that full-length RSV Gag lacking the protease domain is a highly efficient NA chaperone in vitro, and NC is required for this activity. In contrast to results obtained for HIV-1 Gag, due to the weak nucleic acid binding affinity of the RSV MA domain, inositol phosphates do not regulate RSV Gag-facilitated tRNA annealing despite the fact that they bind to MA. These studies provide insight into the viral regulation of tRNA primer annealing, which is a potential target for antiretroviral therapy.

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Section: Discussionsupporting

confidence: 65%

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Rye-McCurdy

Nadaraia-Hoke

Gudleski-O'Regan

et al. 2014

J Virol

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“…In HIV-1, NC plays a major role at this stage and binds the genome specifically. We have recently shown that HIV-1 Gag binds to psi-specific RNAs differently than to non-psi RNAs; our data suggest that psi binding involves primarily the NC domain, whereas non-psi binding involves both the NC and MA domains (58). In contrast, we propose that in deltaretroviruses, MA plays a larger role in recognizing gRNA at the initial stage.…”

Section: Discussioncontrasting

confidence: 55%

Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Sun

Grigsby

Gorelick

et al. 2014

J Virol

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Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro . HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

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“…Hairpins were also extended after exposure to a solution of NC prepared as described previously (33). The specific binding sites on TAR RNA appear to be saturated at 50 nM NC (34,35), as discussed further in SI Materials and Methods, section 9.…”

Section: Methodsmentioning

confidence: 91%

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

McCauley

Rouzina

Manthei

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA-DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA-DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription.single molecule | force spectroscopy | RNA stretching | RNA binding T he transactivation response (TAR) RNA hairpin is a 59-nt sequence in the long-terminal repeat (LTR) of the HIV-1 genome that forms a 24-bp hairpin ( Fig. 1A) (1). This structure is essential in promoting viral transactivator protein (Tat)-mediated transcription. The protein-RNA complex further enhances LTR promoter activity (2). The highly stable TAR hairpin structure that stimulates viral RNA transcription becomes a liability during the early stage of a new infection, as TAR hairpins inhibit the minus-strand transfer step required for reverse transcription (1). To alleviate this inhibition, successful reverse transcription requires a key viral chaperone, the nucleocapsid (NC) protein. In vitro experiments have shown a 3,000-fold stimulation of the rate-limiting step of minus-strand transfer in the presence of NC (3), as NC is required to destabilize TAR RNA and the complementary repeat TAR DNA hairpin to allow subsequent strand annealing (1).HIV-1 NC is only 55 aa long, consisting of two highly conserved CCHC zinc fingers and a basic N terminus (1) (Fig. 1B). The multiple roles of NC during reverse transcription all use the same "chaperone" activity (1), which describes HIV-1 NC's ability to facilitate the rearrangement of nucleic acids into the most stable structures, with the lowest free energy (1). This chaperone activity is characterized by nucleic acid aggregation, duplex destabilization, and rapid kinetics of protein-nucleic acid interactions (3, 4). Aromatic residues in...

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Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Cited by 84 publications

References 79 publications

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Mechanistic Differences between Nucleic Acid Chaperone Activities of the Gag Proteins of Rous Sarcoma Virus and Human Immunodeficiency Virus Type 1 Are Attributed to the MA Domain

Retrovirus-Specific Differences in Matrix and Nucleocapsid Protein-Nucleic Acid Interactions: Implications for Genomic RNA Packaging

Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

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