Single-stranded RNA facilitates nucleocapsid: APOBEC3G complex formation

Abstract

Binding of APOBEC3G to the nucleocapsid (NC) domain of the human immunodeficiency virus (HIV) Gag polyprotein may represent a critical early step in the selective packaging of this antiretroviral factor into HIV virions. Previously, we and others have reported that this interaction is mediated by RNA. Here, we demonstrate that RNA binding by APOBEC3G is key for initiation of APOBEC3G:NC complex formation in vitro. By adding back nucleic acids to purified, RNase-treated APOBEC3G and NC protein preparations in vitro, we demonstrate that complex formation is rescued by short (> or =10 nucleotides) single-stranded RNAs (ssRNAs) containing G residues. In contrast, complex formation is not induced by add-back of short ssRNAs lacking G, by dsRNAs, by ssDNAs, by dsDNAs or by DNA:RNA hybrid molecules. While some highly structured RNA molecules, i.e., tRNAs and rRNAs, failed to rescue APOBEC3G:NC complex formation, other structured RNAs, i.e., human Y RNAs and 7SL RNA, did promote NC binding by APOBEC3G. Together, these results indicate that ternary complex formation requires ssRNA, but suggest this can be presented in the context of an otherwise highly structured RNA molecule. Given previous data arguing that APOBEC3G binds, and edits, ssDNA effectively in vitro, these data may also suggest that APOBEC3G can exist in two different conformational states, with different activities, depending on whether it is bound to ssRNA or ssDNA.

FIGURE 1.

Formation of a specific complex between A3G and HIV-1 NC. (A) 293T cells were transfected with plasmids expressing A3G-Myc-His, GFP-Myc-His, Luc-Myc-His, or A3A-Myc-His. At 44 h after transfection, the cells were lysed and the lysates incubated with recombinant purified GST (lane 1) or GST-NC (lanes 2–5). Bound complexes were then collected using glutathione agarose beads. Input lysates (upper panel, 5% of total) and bound proteins (middle panel, 25% of total) were then visualized by Western analysis using an anti-Myc polyclonal antiserum. GST fusion proteins and non-fused GST were visualized by Western analysis using an anti-GST polyclonal (lower panel). (B) Similar to panel A, except that the 293T cells were transfected with plasmids expressing HA-tagged forms of A3A, A3G, A3G-E1, or A3G-E1+2. GST-NC bound proteins were collected using glutathione agarose beads and input lysates (5% of total) and bound proteins (25% of total) visualized by Western analysis using an HA-specific mouse monoclonal antibody. GST-NC (lower panel) was visualized as described in panel A.

FIGURE 2.

RNase A treatment of A3G blocks formation of an A3G:NC complex. A3G-Myc-His was purified from transfected 293T cells and either left untreated or incubated with RNase A, as indicated. Similarly, GST-NC was purified from bacteria and left untreated or incubated with RNase A. Treated or untreated protein samples were then mixed and incubated together, and any A3G:NC complexes collected using glutathione-agarose beads. The input A3G proteins are shown in the upper panel, lanes 1 and 2, while the bound A3G proteins are shown in lanes 3–6. The GST-NC protein used is shown in the lower panel. Proteins were visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum (upper panel) or an anti-GST polyclonal antibody (lower panel).

FIGURE 3.

Formation of the A3G:NC complex is mediated by unstructured RNAs. (A) The purified A3G-Myc-His was untreated (lanes 3,4) or had been incubated with RNase A (all other lanes). Input A3G-Myc-His protein (5% of total) is shown in lanes 1 and 2. A3G-Myc-His protein was incubated with purified GST (lane 3) or GST-NC (lanes 4–10). In lanes 6–10, yeast RNA was added to the incubation: lane 6, 1.25 μg; lane 7, 2.5 μg; lane 8, 5 μg; lane 9, 10 μg; lane 10, 20 μg. A3G:NC complexes were recovered using glutathione-agarose beads and bound A3G-Myc-His protein visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum (upper panel). Input GST or GST-NC protein was also analyzed by Western blot (lower panel). (B) Similar to panel A, except that the added RNAs (10 μg in each case) represent yeast total RNA (lane 3); purified yeast tRNA (lane 4); total human cell RNA (lane 5), or total human RNA that had been subjected to one round (lane 6) or two rounds (lane 7) of purification on an oligo-dT column. This experiment used exclusively RNase A treated A3G-Myc-His protein. (C) Ethidium bromide-stained agarose gel visualizing 5 μg of each of the RNA samples used in panel B. As may be seen, the yeast RNA sample (Ambion) in lane 1 is degraded to the point where it is smaller than the 70–80-nt-long yeast tRNAs shown in lane 2. Lanes 3–5 reveal the removal of human rRNA as the total human RNA sample (lane 3) was subjected to one (lane 4) or two (lane 5) rounds of oligo-dT purification. (D) Similar to panels A and B, except that this experiment also analyzed in vitro transcribed human Y4 RNA and 7SL RNA.

FIGURE 4.

Formation of A3G:NC complexes is rescued by short, G-containing ssRNAs. (A) Similar to Figure 3B, except that the added RNAs (10 μg) are derived from the fragmented yeast RNA preparation shown in Figure 3C (lane 3) or represent 66 nt (lane 4), 36 nt (lane 5), or 21 nt (lane 6) long ssRNA transcripts generated in vitro using T7 RNA polymerase. (B) Similar to panel A, except that we are here analyzing short, synthetic ssRNAs (0.5 nM). With the exception of random-22 (lane 7), these were all designed to contain only two out the four possible bases and to lack the ability to form duplex molecules. Rescue of A3G:NC complex formation required G residues in the ssRNA (compare lanes 3,4,7 and lanes 5,6) and was more efficient if the oligonucleotide was over 10 nt in length (compare lanes 9,10 and lane 11). Control Western blots analyzing the input level of GST-NC are shown in Supplemental Figure 1.

FIGURE 5.

Rescue of A3G:NC complex formation by ssRNA. (A) Similar to Figure 4B, except that in this experiment the A3G and GST-NC proteins were incubated in the presence of ssRNA oligos (lanes 8,9), ssDNA oligos (lanes 10,11), two complementary ssRNA oligos that had been annealed to form a dsRNA (lane 4), ssRNA oligos that had been annealed to a complementary ssDNA oligo (lanes 5,6) or two complementary ssDNA oligos that had been annealed to form a dsDNA (lane 7) (0.5 nM in each case). Only the G-containing ssRNA oligo (lane 8) and the fragmented yeast RNA positive control (lane 3) rescued complex formation. (B) Similar to Figure 4A, except that this experiment analyzed the ability of 10 μg of denatured salmon testes DNA (lane 4), bacteriophage M13 genomic ssDNA (lane 5), or ssDNA oligonucleotides (lanes 6,7) to rescue formation of the A3G:NC complex. Control Western blots analyzing the input level of GST-NC are shown in Supplemental Figure 1.