Retroviral nucleocapsid proteins and DNA strand transfers

Abstract

An infectious retroviral particle contains 1000-1500 molecules of the nucleocapsid protein (NC) that cover the diploid RNA genome. NC is a small zinc finger protein that possesses nucleic acid chaperone activity that enables NC to rearrange DNA and RNA molecules into the most thermodynamically stable structures usually those containing the maximum number of base pairs. Thanks to the chaperone activity, NC plays an essential role in reverse transcription of the retroviral genome by facilitating the strand transfer reactions of this process. In addition, these reactions are involved in recombination events that can generate multiple drug resistance mutations in the presence of anti-HIV-1 drugs. The strand transfer reactions rely on base pairing of folded DNA/RNA structures. The molecular mechanisms responsible for NC-mediated strand transfer reactions are presented and discussed in this review. Antiretroviral strategies targeting the NC-mediated strand transfer events are also discussed.

Keywords: HIV; Nucleocapsid protein; Retrovirus; Reverse transcription; Strand transfer.

Fig. 1

Sequence and structural features of retroviral NCs from MLV (Genbank #: J02255), HIV-1 (Genbank #: AF324493), HIV-2 (Genbank #: M15390), SIVmne (Genbank #: M32741), HTLV-1 (Genbank #: D13784), HTLV-2 (Genbank #: AF139382), RSV (Genbank #: J02342), MMTV (Genbank #: AF228552), MPMV (Genbank #: M12349).

Fig. 2

Reverse transcription of the retroviral RNA genome. A) The 3′-end of a cellular tRNA (pink line) is annealed to the PBS region of the retroviral RNA (black line). B) Minus-strand DNA (blue line) synthesis is initiated from the tRNA primer to the 5′-end of genome; this product is named minus-strand strong-stop DNA ((−) ssDNA); the genomic RNA is degraded by the RNase H activity of RT. C) The (−) ssDNA is transferred to the 3′-end of the genomic RNA. D) After the first strand transfer, elongation of minus-strand DNA and RNase H degradation continue; plus-strand DNA (red line) synthesis is initiated from a polypurine tract (PPT) sequence that is resistant to RNase H cleavage. E) Elongation of the two DNA strands until the PBS sequences. F) Retroviral RNA, the tRNA and PPT primers are degraded by the RNase H activity of RT. G) The second strand transfer is mediated by base pairing of the complementary PBS sequences at the 3′-ends of minus-strand DNA and plus-strand strong-stop DNA. H) Elongation of minus- and plus-strand DNAs, resulting in a linear double-stranded DNA with a long terminal repeat (U3 R U5) at each end.

Fig. 3

Self-priming of HIV-1 (−) ssDNA. A) Folding of (−) ssDNA into three hairpins ; in the 5′ to 3′ direction, the three shades of blue indicate the u5, cpoly(A) and cTAR domains; the r region is composed of cpoly(A) and cTAR domains. B) Elongation of (−) ssDNA in the presence of RT and dNTPs; the green line indicates the extended DNA strand. The figure is not drawn to scale.

Fig. 4

A possible mechanism for NC-mediated annealing of (−) ssDNA to the 3′-end of the HIV-1 RNA genome. A) (−) ssDNA has been synthesized from the tRNA primer (pink) to the 5′-end of the donor RNA template (DT); the DT (black line) has been degraded by the RNase H activity of RT, a 5′-terminal RNA fragment (14–18 nt) remains annealed at the 3′-end of (−) ssDNA. In the 5′ to 3′ direction, the three shades of blue indicate the u5, cpoly(A) and cTAR domains of (−) ssDNA. B) Formation of two hairpins by the u5 and cpoly(A) domains; the 5′-terminal RNA fragment prevents the formation of the cTAR hairpin. C) NC promotes partial base pairing of the poly(A) hairpin at the 3′-end of the acceptor RNA template (AT) with the complementary cpoly(A) hairpin at the 3′-end of (−) ssDNA. D) The 5′-terminal RNA fragment and small RNA fragments are released in the presence of NC and AT. The figure is not drawn to scale.

Fig. 5

Destabilization of the complementary hairpins by NC. A) TAR RNA destabilization; the red dots indicate the guanines that are key sites for the initiation of destabilization, the black dots indicate the GU wobble base pairs. B) cTAR DNA destabilization; the blue dots indicate the TG mismatched base pairs.

Fig. 6

Proposed initiations sites for NC-mediated annealing of cTAR DNA (blue) to TAR RNA (black). A) Zipper pathway involving initial base pairing of the 3′/5′ termini. B) Loop-loop pathway involving initial formation of an extended loop-loop duplex.

Fig. 7

The acceptor invasion model. A) Minus-strand DNA (blue line) is annealed to the donor RNA template (DT, black line). B) DNA synthesis and RNase H cleavage of the donor RNA template. C) Acceptor docking facilitated by NC; this step allows the acceptor RNA template (AT, gray line) to anneal to the nascent DNA. D) Branch migration facilitated by NC; this step brings AT in proximity to the 3′-end of DNA and allows RNase H-directed cleavages of AT. E) NC-facilitated annealing of AT to the 3′-end of DNA.