Structural and computational studies of HIV-1 RNA

Figures & data

Figure 1. A schematic overview of the HIV-1 replication cycle. The entry of HIV-1 begins with the binding of the HIV-1 virion to the cell membrane through the interactions of the envelope-glycoproteins (Env) with the cell membrane receptors (step 1). This is followed by the fusion of the virion with the cell membrane and entry of the viral particle into the cell (step 2). While the gRNA strands are reverse transcribed into vDNA, the viral core gets transported to the cell nucleus (steps 3–4). vDNA generated through the reverse transcription process is further integrated into the host-cell chromosome (step 5). Upon completion of the integration step, proviral DNA gets transcribed into vRNA molecules which are exported into the cytoplasm (steps 6–7). The vRNA molecules are then either translated into viral proteins or assembled for further packaging with newly synthesized protein molecules (steps 8–9). The assembled viral particle buds on the cell membrane using newly synthesized viral proteins and gRNA and is further released from the cell membrane upon completion of the assembly step (steps 10–11). The release is followed by the maturation process to produce an infectious viral particle (step 12).

Figure 2. The initiation complex of the HIV-1 reverse transcription. Shown are (A) the secondary structures of the PBS-segment RNA and the ${\mathrm{t}\mathrm{R}\mathrm{N}\mathrm{A}}_3^{\mathrm{L}\mathrm{y}\mathrm{s}}$ primer with various structural motifs uniquely colored and labeled; (B) the side-view snapshot of the PBS-segment RNA with key structural motifs uniquely colored and labeled following the color scheme defined in panel A (PDB 7LVA) [47]; and (C) the side-view snapshot of the ARL anticodon of the ${\mathrm{t}\mathrm{R}\mathrm{N}\mathrm{A}}_3^{\mathrm{L}\mathrm{y}\mathrm{s}}$ (pink) annealed to the ARL (blue) of the HIV-1 TLE (light blue; PDB 2K7E) [46]. The modifications in the ARL anticodon motif are highlighted in red. The snapshots in panels B and C utilize the first frame of the corresponding NMR structure. (D) The side-view snapshot of the HIV-1 reverse transcription initiation complex (PDB 6HAK) [48] is shown with two RT domains, the p66 and p51 domains, highlighted in light blue and pink, respectively. The dsRNA structure is colored following the color scheme in panel A. (E) Two conformations of the $3^{\prime }$ end of the anti-PBS segment of the ${\mathrm{t}\mathrm{R}\mathrm{N}\mathrm{A}}_3^{\mathrm{L}\mathrm{y}\mathrm{s}}$ primer annealed to PBS at the RTIC are shown along with the vDNA strand from the elongation complex at the P site (PDBs 6B19, 6HAK, 5TXL) [48–50]. The primer conformations correspond to superimposed functional states of the RTIC on the elongation complex with vDNA: state 1 (dark orange; ${\mathrm{P}}^{\prime }$+1) and state 2 (light orange; ${\mathrm{P}}^{\prime }$) and the elongation complex (white; P).

Table 1. Structural data on the HIV-1 RNA elements participating in reverse transcription. For each structure, shown is the HIV-1 RNA identifier, the corresponding pdb, the binding partner/ligand, the experimental method used to resolve the structure, and the publication year. The following abbreviations are used: ARL, A-rich loop; cryo-EM, cryo-electron microscopy; NCp7, nucleocapsid protein 7; NMR, nuclear magnetic resonance; PBS, primer binding site; RT, reverse transcriptase; TAR, transactivation response element. Furthermore, RNA/DNA is used to indicate the RNA/DNA hybrid.

RNA	PDB	Ligand	Method	Year	Ref.
ARL	2K7E	${\mathrm{t}\mathrm{R}\mathrm{N}\mathrm{A}}_3^{\mathrm{L}\mathrm{y}\mathrm{s}}$	NMR	2009	[46]
TAR/PolyA	7DD4	Apo^†	NMR	2021	[51]
PBS*	2JZW	NCp7	NMR	2008	[52]
PBS	6B19	RT	Cryo-EM	2018	[49]
PBS	6HAK	RT	X-Ray	2019	[48]
PBS	6WAZ	RT	Cryo-EM	2020	[53]
PBS	6WB0	RT	Cryo-EM	2020	[53]
PBS	6WB1	RT	Cryo-EM	2020	[53]
PBS	6WB2	RT	Cryo-EM	2020	[53]
PBS	7KJV	RT	Cryo-EM	2021	[54]
PBS	7KJV	RT/Efavirenz	Cryo-EM	2021	[54]
PBS	7KJX	RT/Nevirapine	Cryo-EM	2021	[54]
PBS^‡	7LVA	Apo	NMR	2021	[47]
RNA/DNA	4PQU	RT/dATP	X-Ray	2014	[55]
RNA/DNA	4PUO	RT/Nevirapine	X-Ray	2014	[55]
RNA/DNA	4PWD	RT/Nevirapine	X-Ray	2014	[55]
RNA/DNA	4Q0B	RT/Nevirapine	X-Ray	2014	[55]

^†Apo indicates the unbound RNA structure.

*The reverse transcribed PBS vDNA structure is reported.

^‡The structure of the full PBS-segment RNA is reported.

Figure 3. TAR RNA and its complexes with various ligands. Shown is (A) the secondary structure of the HIV-1 TAR RNA highlighting various structural motifs (loop, helix 2, bulge, helix 1) that are labeled and colored uniquely; (B) the snapshots of TAR RNA (stick representation) bound to various peptides (left; labeled counterclockwise light orange) and to small-molecules (right; labeled counterclockwise light blue). The ligands are shown in space-filling representations [86–95] with the apo conformation of TAR RNA (PDB 1ANR) shown at the center of the panel [96]; (C) the sequences of the $\beta$2–$\beta$3 loops from four lab-evolved TBPs with key arginine residues highlighted in boxes; (D) the cartoon representation of superimposed TBPs bound to TAR RNA with the $\beta$2–$\beta$3 loops and the key arginine residues and RNA nucleotides (stick representations) highlighted in unique colors following the color scheme in panel C [97,98]; (E) the snapshot of the TBP6.7 binding site with key arginine residues depicted with sticks (PDB 6HX1) [97]; and (F) the snapshot of the G1G truncated joint region between TAR RNA and PolyA RNA along with the truncated secondary structure (PDB 7DD4) [51]. The $5^{\prime }$-terminal G2 and G3 nucleotides and the $3^{\prime }$-terminal UG nucleotides are highlighted in unique colors.

Table 2. Structural data on the HIV-1 RNA elements participating in transcription. Data similar to Table 1 are shown for the HIV-1 RNA elements participating in transcription, and the following additional abbreviations are used: G-quadruplex, guanine-rich quadruplex; SEC, super elongation complex; Tat, transactivator of transcription; TBP, TAR-binding protein.

RNA	PDB	Ligand	Method	Year	Ref.
7SK*	6MCF	Tat	NMR	2018	[99]
G-quadruplex^†	2N4Y	Apo^‡	NMR	2016	[100]
G-quadruplex^†	6H1K	Apo	NMR	2018	[101]
TAR	1ARJ	Arginine amide	NMR	1995	[87]
TAR	1ANR	Apo	NMR	1996	[96]
TAR	1QD3	Neomycin B	NMR	2000	[88]
TAR	1LVJ	Acetylpromazine	NMR	2002	[89]
TAR	1UTS	RBT550	NMR	2004	[90]
TAR	1UUD	RBT203	NMR	2004	[91]
TAR	1UUI	RBT158	NMR	2004	[91]
TAR	2KDQ	L-22	NMR	2009	[92]
TAR	2L8H	MV2003	NMR	2011	[93]
TAR	2KX5	KP-Z-41	NMR	2011	[94]
TAR	5J0M	L-22	NMR	2016	[95]
TAR	5J1O	L-22	NMR	2016	[95]
TAR	5J2W	L-22	NMR	2016	[95]
TAR	6CYT	Tat/SEC	X-Ray	2018	[102]
TAR	6MCE	Tat	NMR	2018	[99]
TAR	6CMN	TBP6.7	X-Ray	2018	[98]
TAR	6D2U	JB181	NMR	2019	[86]
TAR	6XH0	TBP6.9	X-Ray	2020	[97]
TAR	6XH1	TBP6.7	X-Ray	2020	[97]
TAR	6XH2	TBP6.6	X-Ray	2020	[97]
TAR	6XH3	TBP6.3	X-Ray	2020	[97]

*The structure of the human 7SK RNA bound to the HIV-1 Tat protein is reported.

^†G-quadruplex structures of the proviral DNA are reported.

^‡Apo indicates the unbound RNA structure.

Figure 4. Tat protein in complex with 7SK RNA and TAR RNA. Shown are the side-view snapshots of the Tat protein bound to (A) human 7SK RNA (PDB 6MCF) and (B) TAR RNA (PDB 6MCE) [99]. In both snapshots, RNA is shown in a cartoon representation (gray), Tat backbone is shown in a tubular representation (yellow), the ${\mathrm{C}}_{\alpha }$ atoms of those arginine residues which participate in key interactions with both RNA molecules are shown in red spheres, while other arginine residues are shown in blue spheres. The zoomed snapshots correspond to specific interactions formed between the arginine residues in Tat (red sticks) and the RNA nucleotides.

Figure 5. The G-quadruplex conformation from LTR-3. Two snapshots of a G-quadruplex DNA structure from the LTR-3 along with the sequence of the LTR-3 domain are shown. DNA is depicted in a ribbon representation (gray); guanine, adenine, thymine, and cytosine bases are depicted in cyan, green, orange, and red stick representations, respectively. A pink arrow in the right panel follows the V-shape of the $3^{\prime }$-end loop.

Table 3. Structural data on the HIV-1 RNA elements participating in nuclear export. Data similar to Table 1 are shown for the HIV-1 RNA elements participating in nuclear export and the following additional abbreviations are used: Rev, regulator of expression of virion; RRE, Rev response element.

RNA	PDB	Ligand	Method	Year	Ref.
RRE	1ETF	Rev	NMR	1996	[124]
RRE	1CSL	Apo^a	X-Ray	2000	[125]
RRE	1DUQ	Apo	X-Ray	2000	[126]
RRE	1G70	RSG-1.2	NMR	2001	[127]
RRE	1I9F	RSG-1.2	NMR	2001	[128]
RRE	4PMI	Rev dimer	X-Ray	2014	[129]

^aApo indicates the unbound RNA structure.

Figure 6. RRE-IIB RNA in complex with the helical peptides. Shown are (A) the sequences of the HIV-1 RRE-IIB RNA, the Rev peptide, and the RSG-1.2 peptide with key nucleotides and amino acids highlighted in blue and red, respectively; (B) the snapshots of the HIV-1 RRE-IIB RNA bound to Rev peptide (PDB 1ETF) [124]; and (C) the snapshots of the HIV-1 RRE-2B RNA bound to the RSG-1.2 peptide (PDB 1G70) [127]. In all snapshots, RNA is shown in a cartoon representation (light green) with the blue spheres corresponding to phosphorous atoms for those nucleotides which form crucial interactions with the peptides and with the green spheres corresponding to phosphorous atoms in the backbone of other RNA nucleotides; the peptide backbone is shown in a tubular representation (white) with red spheres corresponding to the ${\mathrm{C}}_{\alpha }$ atoms of key arginine residues. The snapshots of the entire RNA/peptide complexes are accompanied with snapshots highlighting the corresponding interactions between key arginine amino acids and RNA nucleotides which are represented as red and blue sticks, respectively.

Figure 7. Structural features of the dimerization process. Shown are (A) the scheme and secondary structure of the dimerization process; (B) the snapshot of the overall kissing complex and the zoomed-in view of the kissing loop region (PDB 2D1B) [183]; and (C) the example snapshots of two extended duplexes which display different configurations of the bulged nucleotides (PDBs 462D and 2D1A) [183,185]. The zoomed-in view on one of the bulged motifs is provided for the second extended duplex. In each panel, nucleotides of the DIS kissing loops (G274 to C279) participating in the recognition process are highlighted in darker colors.

Table 4. Structural data on the HIV-1 RNA elements participating in the gRNA dimerization. Data similar to Table 1 are shown for the HIV-1 RNA elements participating in the gRNA dimerization and the following additional abbreviations are used: BM, bulge-out motif; DIS, dimerization initiation site; ED, extended duplex; KL, kissing loop; MD, molecular dynamics simulation; SL, stem-loop. Asterisk (*) indicates structures containing DNA.

RNA	PDB	Ligand	Method	Year	Ref.
DIS	2N1Q	Apo	NMR	2015	[179]
DIS BM	1M5L	Apo	NMR	2002	[180]
DIS BM	1N8X	Apo	NMR	2003	[181]
DIS BM	1OSW	Apo	NMR	2003	[182]
DIS BM	2D17	Apo	NMR	2005	[183]
DIS ED	1JU1	Apo	NMR	1999	[184]
DIS ED	462D	Apo	X-Ray	1999	[185]
DIS ED	1NLC	Zn^2+	X-Ray	2003	[186]
DIS ED	1O3Z	Hexamine	X-Ray	2003	[186]
DIS ED	1WVD	CoCl2	X-Ray	2003	[186]
DIS ED	1Y6S	Ba^2+	X-Ray	2003	[186]
DIS ED	1Y6T	Co^3+/Hexamine	X-Ray	2003	[186]
DIS ED	1Y73	Pt^4+	X-Ray	2003	[186]
DIS ED	1Y90	Mn^2+	X-Ray	2003	[186]
DIS ED	1Y95	Pb^2+	X-Ray	2003	[186]
DIS ED	2OIJ	AuCl3	X-Ray	2003	[186]
DIS ED	2D18	Apo	NMR	2005	[183]
DIS ED	2D1A	Apo	NMR	2005	[183]
DIS ED	2QEK	Apo	X-Ray	2006	[187]
DIS ED	2GM0	Apo	NMR	2006	[188]
DIS ED	3C3Z	Ribostamycin	X-Ray	2008	[187]
DIS ED	3C44	Paromomycin	X-Ray	2008	[187]
DIS ED	3C5D	Lividomycin	X-Ray	2008	[187]
DIS ED	3C7R	Neomycin	X-Ray	2008	[187]
DIS ED	2OIY	Apo	X-Ray	2010	[189]
DIS ED	2OJ0	Apo	X-Ray	2010	[189]
DIS ED	6BG9	Apo	Cryo-EM/NMR/MD	2018	[190]
DIS KL	1BAU	Apo	NMR	1998	[191]
DIS KL	1K9W	Apo	X-Ray	2001	[192]
DIS KL	2B8R	Apo	X-Ray	2001	[192]
DIS KL	2B8S	Apo	X-Ray	2001	[192]
DIS KL*	1JU0	Apo	NMR	2002	[193]
DIS KL*	1JUA	Apo	NMR	2002	[193]
DIS KL	2D19	Apo	NMR	2005	[183]
DIS KL	2D1B	Apo	NMR	2005	[183]
DIS KL	2F4X	Apo	NMR	2006	[194]
DIS KL	1XP7	Apo	X-Ray	2006	[195]
DIS KL	1XPE	Apo	X-Ray	2006	[195]
DIS KL	1XPF	Apo	X-Ray	2006	[195]
DIS KL	1Y3O	Mn^2+	X-Ray	2006	[195]
DIS KL	1Y3S	Apo	X-Ray	2006	[195]
DIS KL	1YXP	Zn^2+	X-Ray	2006	[195]
DIS KL	1ZCI	Apo	X-Ray	2006	[195]
DIS KL	2FCX	Neamine	X-Ray	2006	[196]
DIS KL	2FCY	Neomycin	X-Ray	2006	[196]
DIS KL	2FCZ	Ribostamycin	X-Ray	2006	[196]
DIS KL	2FD0	Lividomycin	X-Ray	2006	[196]
DIS SL	1JTJ	Apo	NMR	2002	[197]
SL2	1F6U	NCp7	NMR	2000	[198]
SL3	1A1T	NCp7	NMR	1998	[199]
TAR/PolyA/U5	6VVJ	Apo	NMR	2020	[114]
TAR	6VU1	Apo	NMR	2020	[114]

Table 5. A summary of simulation studies on the HIV-1 RNA. For each simulation study, listed is the simulated HIV-1 RNA element, corresponding simulation conditions, and the force-field used in RNA simulations. The cumulative simulation time-scale, the temperature, and simulation methods are also listed for each study. The RNA force-fields summarized are CHARMM27 [267], CHARMM36 [268], Cornell et al. [269], parm98 [270], parmbsc0 [271], ff99OL3 [271–273], and RNA.ROC [274]. The missing simulation parameters or force-field details in the table is due to the absence of such information in some simulation studies [258,275–278]. The following abbreviations are used: CG, coarse-grained; DFT, density functional theory; FEP, free energy perturbation; KL, kissing loop; MD, molecular dynamics; MM-GBSA, molecular mechanics with generalized born and surface area solvation; PBE, Poisson–Boltzmann equation; QM, quantum mechanics-based reparameterization of the AMBER force field; RAM, replica-averaged metadynamics; SL1, stem loop 1; SMD, steered molecular dynamics. Furthermore, RNA/DNA indicates the structure containing RNA/DNA hybrid.

#	RNA	Time (ns)	T (K)	Method	Force-field	Ref.
1	DIS SL1	10	300	MD	parm98	[197]
2	DIS KL	29.4	300	MD	Cornell et al.	[279]
3	DIS KL	800	300	MD	CHARMM27	[280]
					parmbsc0
4	DIS KL	25000	300	MD	ff99OL3	[281]
5	RNA/DNA	300	—	MD	CHARMM27	[275]
6	RNA/DNA	5200	—	MD	ff99OL3	[276]
7	RNA/DNA	5200	—	MD	ff99OL3	[277]
8	RRE	100	300	MD	parm98	[282]
9	RRE	3900	310	MD/SMD	RNA.ROC	[151]
10	RRE	50000	310	MD/FEP	CHARMM36	[147]
11	TAR	2	300	MD/PBE	Cornell et al.	[283]
12	TAR	2	298	MD/MM-GBSA	Cornell et al.	[284]
13	TAR	200	300	MD/MM-GBSA	parm98	[285]
14	TAR	80	300	MD	CHARMM27	[286]
15	TAR	100	300	MD	parm98	[282]
16	TAR	10	300	MD/MM-GBSA	QM	[287]
				DFT
17	TAR	65	300	MD	CHARMM27	[288]
17	TAR	200	300	MD	Cornell et al.	[289]
19	TAR	500	300	MD	parmbsc0	[111]
20	TAR	8200	300	MD	CHARMM36	[290]
21	TAR	4000	300	MD	parmbsc0	[291]
22	TAR	4200	300	MD/SMD	ff99SB-ILDN	[292]
				PBE
23	TAR	80	300	MD/MM-GBSA	parmbsc0	[293]
24	TAR	800	—	MD	ff99OL3	[278]
25	TAR	640	298.15	MD/RAM	ff99OL3	[95]
26	TAR	60	300	MD/MM-GBSA	—	[258]
27	TAR	20	—	MD	ff99SB-ILDN	[294]
28	TAR	20	—	MD/MM-GBSA	ff99SB-ILDN	[295]
29	TAR	16400	300	MD/FEP	CHARMM36	[277]
30	TAR	—	298–383	CG	CG potential	[296]
31	TAR	2500	310	MD/SMD	ff99OL3	[297]
32	TAR	54000	300	MD	ff99OL3	[106]
33	TAR	16400	300	MD/FEP	CHARMM36	[113]
34	TAR	14000	300	MD/SMD	parmbsc0	[298]

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