Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription

Abstract

The HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which remodels nucleic acid structures so that the most thermodynamically stable conformations are formed. This activity is essential for virus replication and has a critical role in mediating highly specific and efficient reverse transcription. NC's function in this process depends upon three properties: (1) ability to aggregate nucleic acids; (2) moderate duplex destabilization activity; and (3) rapid on-off binding kinetics. Here, we present a detailed molecular analysis of the individual events that occur during viral DNA synthesis and show how NC's properties are important for almost every step in the pathway. Finally, we also review biological aspects of reverse transcription during infection and the interplay between NC, reverse transcriptase, and human APOBEC3G, an HIV-1 restriction factor that inhibits reverse transcription and virus replication in the absence of the HIV-1 Vif protein.

Figure 1

Primary sequence of HIV -1 NC and nucleic acid structures required for the strand transfer steps in reverse transcription. (A) HIV-1 NC. The sequence shown is for the NL4-3 isolate (GenBank accession no. AF324493). The zinc coordinating residues are shown in red and the basic residues are highlighted in blue. (B) Structures of TAR RNA, TAR DNA, (−) PBS and (+) PBS. The NL4-3 TAR RNA structure is based on the structures described in reference and . TAR DNA is shown here as the complement of the TAR RNA structure, but it can also form alternative conformations (reviewed in ref. 146). The PBS structures are based on the structures given in reference and .

Figure 2

Schematic diagram of events in reverse transcription. Step 1. Reverse transcription is initiated by the cellular tRNA primer (tRNA^Lys₃). The tRNA primer is represented by a short orange line attached to a “clover-leaf” (remaining tRNA bases). The 3′ 18 nt of the primer are annealed to an 18 nt sequence in vRNA (red solid line; upper case lettering) known as the PBS, which is located at the 5′ end of the genome. This reaction is termed “tRNA primer placement”. Step 2. RT catalyzes extension of the primer to form (−) SSDNA (blue solid line; lower case lettering), which contains copies of the unique 5′ genomic sequence (U5) and R regions. As the primer is extended, the RNase H activity of RT degrades the vRNA sequences (fragments represented by short red solid lines) that have already been reverse transcribed. Step 3. To elongate the minus-strand, (−) SSDNA is transferred to the 3′-end of the genome, which is facilitated by annealing of the complementary R sequences. This step is termed minus-strand transfer. Step 4. Following minus-strand transfer, elongation of minus-strand DNA and RNase H degradation continue. Plus-strand DNA synthesis is initiated by the 15 nt 3′ PPT, located immediately upstream of the unique 3′ genomic sequence (U3). A second 15 nt PPT (cPPT), with the same sequence as the 3′ PPT, is located at the center of the genome in the IN coding region (not shown here). The role of the cPPT in plus-strand synthesis is illustrated in Figure 5. The PPT duplexes are resistant to RNase H cleavage. Step 5. RT copies the u3, r and u5 regions in minus-strand DNA as well as the 3′ 18 nt of the tRNA primer, thereby reconstituting the PBS. The product formed is referred to as (+) SSDNA. Step 6. The tRNA and PPT primers are eventually removed by RNase H from minus- and plus-strand DNAs, respectively. Step 7. Plus-strand transfer is facilitated by annealing of the complementary PBS sequences at the 3′ ends of (+) SSDNA and minus-strand DNA. This is followed by circularization of the two DNA strands and displacement synthesis to generate two LTR sequences. Step 8. Minus- and plus-strand DNAs are elongated, resulting in a linear, dsDNA with an LTR at each end. This DNA is integration-competent. Adapted from reference and with permission from Elsevier and Oxford Journals (Oxford University Press), respectively.

Figure 3

Secondary structure model generated by SHAPE-constrained folding of the ex virio state of the first 578 nt of the HIV-1 vRNA genome (adapted from ref. 55). Nucleotides proposed to form intermolecular bp with a second monomer are enclosed within a black box; the AUG start codon for the Gag polyprotein (nt 336) is highlighted in bold. Nucleotides are colored according to their SHAPE reactivity; bars indicating base pairing are colored by their “pairing persistence”, as defined in reference . Effects of pretreatment of viral particles with AT-2, a zinc ejector that disrupts NC-RNA interactions (see text), are indicated with filled (strong reactivity) and clear (weak reactivity) arrowheads. Clustered sites that show a strong increase in reactivity following treatment with AT-2 and report specific NC binding sites are highlighted in cyan; proposed primary and secondary sites are identified with double (**) and single asterisks (*), respectively. Sites within the first 185 nt that show very strong reductions in SHAPE reactivity following AT-2 treatment, thus reporting on the destabilizing activity of NC, are indicated by solid gray arrows. Additional sites at well-spaced intervals in the vRNA that also display enhanced SHAPE reactivity upon compromising the zinc-binding motif are indicated by dashed gray arrows. The primer is tRNA^Lys₃ shown starting at nt 33 bound to the vRNA via the anticodon domain and variable arm.

Figure 4

Schematic representation of “Roadblock” mechanism to explain the inhibition of RT-mediated primer extension by A3G or Gag. (A) Rapid on-off binding kinetics displayed by NC upon binding to the ss nucleic acid template allows primer extension by RT to proceed. (B) Slow dissociation of A3G or Gag from the nucleic acid template blocks RT movement along the template during primer extension. The presence of A3G or Gag results in reduced primer extension (B) compared to extension in the presence of NC (A). The gray dashes followed by an arrow represent extension from the primer (solid gray). RT is depicted as a purple ellipse and the template is shown as a solid black line. NC and Gag or A3G are highlighted in green and red, respectively.

Figure 5

cPPT priming and formation of the central flap in plus-strand DNA synthesis. Step 1. Plus-strand DNA synthesis is initiated from two identical RNA primers: cPPT and 3′ PPT (shown in blue). The template consists of minus-strand DNA (in black) attached to tRNA (in green) at its 5′-end. The short segments of plus-strand DNA extended from both primers are shown in red. Step 2. RT extends the 3′PPT primer until the 3′ 18-nt of the tRNA (complement of PBS) is copied leading to the formation of (+) SSDNA with a reconstituted PBS sequence (also see Fig. 2). Concomitant with extension, RNase H removes the two PPT primers. With the assistance of NC, RNase H also removes the tRNA primer so that plus-strand synthesis may proceed. Plus-strand transfer occurs when the PBS sequence at the 3′-end of (+) SSDNA anneals to the PBS sequence at the 3′-end of minus-strand DNA. This leads to circularization of vDNA. Strand displacement synthesis of minus-strand DNA begins (see step 3). Extension of plus-strand DNA originating from the cPPT primer is also shown. Step 3. Left side. Minus-strand DNA is extended (shown by black arrow) by copying the (+) SSDNA sequence and in the process, the 5′-end of minus-strand DNA (originally annealed to (+) SSDNA) is displaced. (+) SSDNA is extended using the minus-strand DNA template until the cPPT sequence is copied. Further elongation proceeds by strand displacement of plus-strand DNA primed by the cPPT primer. Termination occurs when the end of the CTS is reached. This results in the formation of a triple-stranded structure containing a flapping strand (flap) in the middle of the dsDNA product (see step 4). Right side. Elongation of minus-strand DNA (black arrow) and plus-strand DNA (from the cPPT primer) (red arrow) by strand displacement synthesis continues. Step 4. This process ultimately leads to formation of LTR (U3-R-U5) regions at both ends of the linear dsDNA product. The flap region and gap shown at the center of the dsDNA are thought to be repaired by cellular enzymes.

Figure 6

Effect of A3G on HIV -1 replication. During virus assembly at the plasma membrane of the producer cell, packaging of A3G proteins into newly formed virions occurs in the absence of the HIV-1 Vif protein. A3G-containing virions enter the target cell (on the right) and undergo reverse transcription. During this process, the deaminase activity of A3G causes C to U mutations on exposed ssDNA regions generated during reverse transcription (deaminase-dependent mechanism). These mutations are detrimental to the virus and therefore lead to inhibition of virus replication. A3G may also interfere with virus replication in a deaminase-independent manner (no C to U mutations) by acting as a “roadblock” to the movement of RT during the primer extension step (see Fig. 4). Adapted from reference with permission from the Royal Society (UK).