Advances in understanding the initiation of HIV-1 reverse transcription

Abstract

Many viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Human Immunodeficiency Virus (HIV), use RNA as their genetic material. How viruses harness RNA structure and RNA-protein interactions to control their replication remains obscure. Recent advances in the characterization of HIV-1 reverse transcriptase, the enzyme that converts its single-stranded RNA genome into a double-stranded DNA copy, reveal how the reverse transcription complex evolves during initiation. Here we highlight these advances in HIV-1 structural biology and discuss how they are furthering our understanding of HIV and related ribonucleoprotein complexes implicated in viral disease.

Figure 1

Overview of reverse transcription mechanism. Reverse transcription with tRNA^Lys₃ annealing to the viral RNA (1). RT initiates with minus strand cDNA synthesis until it reaches the repetitive region R at the 5′ end of the vRNA template (2). The first strand transfer occurs, resulting in the extended primer annealed to the complementary repetitive region R at the 3′ end of the template (3). cDNA synthesis proceeds again and the vRNA template is degraded except for the ppt (4). Plus-strand synthesis begins with RT using the remaining ppt track as a primer (5) and extending the primer until it reaches the PBS region (6). All remaining RNA is degraded (7) and the complementary PBS sequences are used to facilitate the second strand transfer so plus strand cDNA synthesis can continue (8). The PBS region of the minus strand is extended to copy the U3, R, and U5 regions (9). The final product is double stranded DNA with U3, R, and U5 regions flanking the protein coding region of the genome.

Figure 2

The structure of RTIC. (a) The RTIC cryoEM map by Larsen et al. [13^••] reveals density of the vRNA/tRNA PBS duplex beyond the nucleic acid binding cleft within the RT (PDB ID: 6B19). The p66 and p51 subunits are depicted in purple and grey. RT is bound to a vRNA/tRNA duplex depicted in gold and maroon. (b) In addition to the PBS duplex accommodated within the RT, the vRNA forms two helixes H1 and H2, while the tRNA forms an extended helix conformation, shown in RTIC schematic representation of the secondary structure. (c) The polymerase active site is further divided into the fingers, thumb, palm, and connection subdomains. Encircling RT’s active site is the canonical reverse transcription catalysis cycle. (d) Helical geometries are shown for DNA/DNA (PDB ID: 4C64, [65]), DNA/RNA (PDB ID: 1EFS, [66]), and RNA/RNA (PDB ID: 1RNA, [67]), representative of the duplexes involved in reverse transcription.

Figure 3

RT complexes in initiation and elongation, focusing on the core. (a) Both RTIC structures have open fingers and hyperextended thumb, Larsen et al. [13^••] cryoEM in red (PDB ID: 6B19) and Das et al. [15^••] crystal structure in green (PDB ID: 6HAK), compared to an elongation complex solved by Huang et al. [68] that is an RT-DNA/DNA structure in pink (PDB ID: 1RTD). (b) The 3′ termini in initiation structures are displaced by ∼6 Å from the active site with respect to the elongation complex; same color scheme as above (c) Top view of RNase H active site shows that template and primer are flexible to engage with the RNase H region in different RT-nucleic acid complexes; in the Tian et al. [17^••] structure, RNA is in green and DNA is in cyan (PDB ID: 6BSH), compared to Larsen et al. [13^••] structure where vRNA is shown in yellow and tRNA shown in red (PDB ID: 6B19). (d) Closeup view of the RNase H region demonstrates how the RNase H grip structure is pushing the DNA strand away and the RNA strand is pulled into the active site in an active DNA–RNA hybrid (PBD ID: 6BSH), while the vRNA and tRNA remain rigid and do not engage with the RNase H region in the RTIC (PBD ID: 6B19).

Figure 4

vRNA structure modulation by RT binding at initiation. (a) Schematics for smFRET experimental setup to measure the H1 helix formation by fluorescently labeling the vRNA construct at 3′ and 5′ of H1. Histograms of population densities show that H1 (high FRET) is stabilized in presence of RT (adopted from Coey et al. [34^••]). (b) Schematics for smFRET experimental setup to measure the global fold of the initiation complex, with vRNA construct labeled at 5′ of H1 and at the 5′ of tRNA. Histograms of population densities show that upon RT binding the global conformation shifted towards a lower FRET state, indicating a stabilization of a global conformation for the initiation complex. (c) Schematics for smFRET experimental setup to measure the global fold of the initiation complex, switching the vRNA construct label to the 3′ of H1. The results using this labeling scheme showed again that upon RT binding there is a stabilization of the initiation complex formation.