HIV-1 reverse transcription

Abstract

Reverse transcription and integration are the defining features of the Retroviridae; the common name "retrovirus" derives from the fact that these viruses use a virally encoded enzyme, reverse transcriptase (RT), to convert their RNA genomes into DNA. Reverse transcription is an essential step in retroviral replication. This article presents an overview of reverse transcription, briefly describes the structure and function of RT, provides an introduction to some of the cellular and viral factors that can affect reverse transcription, and discusses fidelity and recombination, two processes in which reverse transcription plays an important role. In keeping with the theme of the collection, the emphasis is on HIV-1 and HIV-1 RT.

Figure 1.

Conversion of the single-stranded RNA genome of a retrovirus into double-stranded DNA. (A) The RNA genome of a retrovirus (light blue) with a tRNA primer base paired near the 5′ end. (B) RT has initiated reverse transcription, generating minus-strand DNA (dark blue), and the RNase H activity of RT has degraded the RNA template (dashed line). (C) Minus-strand transfer has occurred between the R sequences at both ends of the genome (see text), allowing minus-strand DNA synthesis to continue (D), accompanied by RNA degradation. A purine-rich sequence (ppt), adjacent to U3, is resistant to RNase H cleavage and serves as the primer for the synthesis of plus-strand DNA (E). Plus-strand synthesis continues until the first 18 nucleotides of the tRNA are copied, allowing RNase H cleavage to remove the tRNA primer. Most retroviruses remove the entire tRNA; the RNase H of HIV-1 RT leaves the rA from the 3′ end of the tRNA attached to minus-strand DNA. Removal of the tRNA primer sets the stage for the second (plus-strand) transfer (F); extension of the plus and minus strands leads to the synthesis of the complete double-stranded linear viral DNA (G).

Figure 2.

Structure of a ternary complex of HIV-1 RT, double-stranded DNA, and an incoming dNTP. HIV-1 RT is composed of two subunits, p51 and p66. P51 is shown in gray. The RNase H domain of p66 is gold, and the four subdomains of the polymerase domain of p66 are color-coded: fingers, blue; palm, red; thumb, green; and connection, yellow. The template strand of the DNA is brown, and the primer strand is purple. The incoming dNTP is light blue. (Figure courtesy of K. Das and E. Arnold.)

Figure 3.

Structural changes in RT that occur during polymerization. In unliganded RT (A), the thumb is in the closed configuration. Binding a double-stranded nucleic acid substrate (B) is accompanied by movement of the thumb (upper left, A,B) that creates the nucleic acid binding site. Binding of the incoming dNTP (C) is accompanied by a movement of the fingers that closes the β3-β4 loop down onto the incoming dNTP (lower left, B,C). These movements correspond to steps in DNA synthesis (bottom). (Figure courtesy of K. Kirby and S. Sarafianos.)

Figure 4.

Recombination models. (A) Copy-choice model, (B) dynamic copy-choice model. The top of A shows two RNA strands (thin orange and green lines). Minus-strand DNA synthesis uses the green RNA strand as a template; however, the green strand is nicked, causing DNA synthesis to switch to the orange RNA strand. This switch leads to the generation of a double-stranded DNA that is composed of sequences from both the green and the orange RNAs. In B, minus-strand DNA synthesis also uses the green RNA strand as a template. When the viral DNA is synthesized, RNase H degrades the green RNA strand, and the DNA that was copied from the green RNA strand can hybridize to the orange RNA (middle panels). This facilitates a transfer of the growing minus-strand DNA from the green to the orange strand, which results in the synthesis of a double-stranded DNA with sequences from both of the parental RNAs.