Diverse interactions of retroviral Gag proteins with RNAs

Abstract

Retrovirus particles are constructed from a single virus-encoded protein, termed Gag. Given that assembly is an essential step in the viral replication cycle, it is a potential target for antiviral therapy. However, such an approach has not yet been exploited because of the lack of fundamental knowledge concerning the structures and interactions responsible for assembly. Assembling an infectious particle entails a remarkably diverse array of interactions, both specific and nonspecific, between Gag proteins and RNAs. These interactions are essential for the construction of the particle, for packaging of the viral RNA into the particle, and for placement of the primer for viral DNA synthesis. Recent results have provided some new insights into each of these interactions. In the case of HIV-1 Gag, it is clear that more than one domain of the protein contributes to Gag-RNA interaction.

Box 1, Figure I

(a) A transmission electron micrograph of a section of a human cell expressing HIV-1 Gag and includes a single immature virus particle being released from the cell. (b) Gag is a multi-domain protein always containing, from N- to C-terminus, the MA domain (shown in green), the CA domain (blue), and the NC domain (red). Depending on the genus of retrovirus, one or a few additional domains or spacers may also be present. (c) The radial arrangement of Gag molecules that are in contact with membrane on the outside and RNA (yellow line) on the inside of the immature particle. There are on the order of 7 nucleotides per Gag molecule in the particle. Panel a is reproduced, with permission from [xxx].

Fig. 1. Proposed mechanism of dependence of virus particle assembly on nucleic acid

Purified Gag proteins are soluble, but assemble into virus-like particles when NA is added. The fact that short oligonucleotides (~ 30 bases) can support assembly suggests that oligomerization renders the Gag assembly-competent. The available data suggest that when several Gag molecules are in close proximity, the very high local Gag concentration induces a conformational change, leading to the exposure of new interfaces (shown here in the C-terminal portion of the CA domain; blue) for protein–protein interactions, and in turn to virus particle assembly. In HIV-1, a high concentration appears to induce a helical conformation in SP1, a short “spacer” between NC (red) and CA [18]. This model was originally suggested by Ma and Vogt [8]. MA domain: green.

Fig. 2. HIV-1 Gag undergoes major conformational changes during virus particle assembly

HIV-1 Gag protein assumes compact conformations in solution, with both ends near each other in 3-dimensional space (i ) [22]. Both the N-terminal, MA domain (green) and the C-terminal, NC domain (red) are positively charged, but the MA domain (blue) has a much higher affinity for negatively charged phospholipids than for RNA; the NC domain exhibits the converse pattern. Nonspecific electrostatic interactions maintain the compact conformations of the protein when it is bound to membrane with negatively charged phospholipids (ii), or when bound to RNA (iv). However, when both types of ligands are present, the protein extends to the rod-shaped conformation present in authentic virus particles (iii) (see Box 1)[24].

Fig. 3. The MLV packaging signal contains a riboswitch

All retroviral vRNAs are packaged into virus particles in dimeric form. In MLV, the contacts between the two monomers in the dimer (ii) include the extended base-paired stretches “PAL1” and “PAL2” (green and red boxes), and “kissing-loop” interactions between SL1 in one monomer and SL2 in the other (orange boxes). Each monomer contains two UCUG-UPu-UCUG motifs (blue boxes). Dimerization involves a “switch”, in that the UCUG-UPu-UCUG motifs are partially or fully base-paired in the monomer (i), but unpaired in the dimer. They are high-affinity binding sites for MLV Gag protein and are bound by NC protein in mature MLV particles; replacement of the four G residues with A residues prevents selective packaging of vRNA [50-52].

Figure 4. Substrates for Gag-facilitated RNA refolding events that occur during HIV-1 assembly

HIV-1 Gag catalyzes annealing of the primer tRNA^Lys3 (ii) to complementary regions on the viral genome (i). Three regions of tRNA^Lys3 (1, 2, 3, red) interact with 3 complementary regions on the genome (1*, 2*, 3*, red). The highly conserved 18-nt PBS (1*) is complementary to the 3′ 18 nt (1) of tRNA^Lys3. Reverse transcription initiates from the 3′-hydroxyl of A76. Regions 2* and 3* have been proposed to interact with the anticodon stem and variable arm (2) and anticodon loop (3), respectively, but their roles in tRNA^Lys3 priming are not as well established. The 2/2* interaction is important for efficient initiation of cDNA synthesis [83], and a similar interaction occurs in RSV [84]. The 3/3* interaction between the U-rich tRNA^Lys3 anticodon and A-rich bulge upstream of the PBS has been proposed to help stabilize the tRNA^Lys3 primer [85]. These interactions are consistent with SHAPE analysis of the genome [53].