Functional analysis of the core human immunodeficiency virus type 1 packaging signal in a permissive cell line

Abstract

Packaging of type C retrovirus genomic RNAs into budding virions requires a highly specific interaction between the viral Gag precursor and unique cis-acting packaging signals on the full-length RNA genome, allowing the selection of this RNA species from among a pool of spliced viral RNAs and similar cellular RNAs. This process is thought to involve RNA secondary and tertiary structural motifs since there is little conservation of the primary sequence of this region between retroviruses. To confirm RNA secondary structures, which we and others have predicted for this region, disruptive, compensatory, and deletion mutations were introduced into proviral constructs, which were then assayed in a permissive cell line. Disruption of either of two predicted stem-loops was found to greatly reduce RNA encapsidation and replication, whereas compensatory mutations restoring base pairing to these stem-loops had a wild-type phenotype. A GGNGR motif was identified in the loops of three hairpins in this region. Results were consistent with the hypothesis that the process of efficient RNA encapsidation is linked to dimerization. Replication and encapsidation were shown to occur at a reduced rate in the absence of the previously described kissing hairpin motif.

FIG. 1

Nucleic acid sequences of mutants created by introducing mutations into the HIV-1 HXB2 provirus. Altered bases are underlined. The positions of deletions are indicated by vertical lines. The protein sequence of the D4 mutant is shown below the primary sequence, and the glycine-to-leucine change is underlined.

FIG. 2

Positions of mutations within the RNA secondary structures of the HIV-1 HXB2 5′ leader sequence showing our original prediction for the structure of SL2 (stem 2), which differs from that of Sakaguchi et al. (57). The sequence deleted in the ΔP2 mutation is shaded. Abbreviations: del, deletion: disr, disruption: comp, compensatory mutation. Stems 1 to 4, SL1 to SL4.

FIG. 3

(A) Predicted sizes (in bases) of the protected RNA fragments for the WT sequence of the HIV-1 HXB2 provirus after RNase protection assays. LTR, long terminal repeat. (B) Predicted sizes (in bases) of the protected RNA fragments which characterize mutant sequences after RNase protection assays using the WT riboprobe.

FIG. 3

(A) Predicted sizes (in bases) of the protected RNA fragments for the WT sequence of the HIV-1 HXB2 provirus after RNase protection assays. LTR, long terminal repeat. (B) Predicted sizes (in bases) of the protected RNA fragments which characterize mutant sequences after RNase protection assays using the WT riboprobe.

FIG. 4

Western blots of proteins extracted from cytoplasms (a, c, and e) and supernatants (b, d, and f) of Jurkat-tat cells transfected with the WT and mutant HIV-1 proviruses A1 to A3 and A5 to A13, probed with a monoclonal antibody to p55/24. Mock, nontransfected cells.

FIG. 5

Western blots of proteins extracted from the cytoplasms (a, c, and e) and supernatants (b, d, and f) of Jurkat-tat cells, transfected with the WT and mutant HIV-1 proviruses A1 to A3 and A5 to A13, probed with a monoclonal antibody to gp120/160. Mock, nontransfected cells.

FIG. 6

Western blots of proteins extracted from the cytoplasms (a and c) and supernatants (b and d) of Jurkat-tat cells transfected with the WT and mutant HIV-1 proviruses D1 to D4, probed with a monoclonal antibody to p55/24 (a and b) or to gp120/160 (c and d). Mock, nontransfected cells.

FIG. 7

RT activity in the supernatants of Jurkat-tat cells infected with the WT and mutant proviruses (counts per second of [³⁵S]TTP incorporation per 10 μl) plotted against days postinfection. (A) Mutations to SL1; (B) mutations to SL2; (C) mutations to SL3; (D) mutations to purine motifs.

FIG. 8

RPAs of RNAs extracted from virions in the supernatants of chronically infected Jurkat-tat cells. Abbreviations: M, RNA size markers; P, undigested riboprobe; D, digested riboprobe; U, uninfected cells; LTR, long terminal repeat. Lanes containing relevant mutants are indicated.

FIG. 9

RPAs of RNAs extracted from the cytoplasms of chronically infected Jurkat-tat cells. Abbreviations: M, RNA size markers; P, undigested riboprobe; D, digested riboprobe; U, uninfected cells; LTR, long terminal repeat. Lanes containing relevant mutants are indicated.

FIG. 10

Intensity of protected RNA bands, after RPAs with virion RNAs, shown as a percentage of the intensity of the band from WT RNA.

FIG. 11

RNA secondary-structure predictions done by use of the GCG program MFold. Bases which differ from those of HIV-1 HXB2 are in lowercase letters. (a) The divergent HIV-1 isolate ANT70C. Bases in MVP5180 which differ from those of ANT70C are shown in boxes. (b) CIV.

FIG. 12

Predictions of RNA secondary structures within SL2 of RNAs from mutant proviruses, based on the structure for this region first documented by Sakaguchi et al. (57), by using the output program MFold. Predictions of similar structures in the equivalent regions of the HIV-1 isolates RF, Z2Z6, ANT70C, and HIV-2 ROD are shown.