5′ leader region of HIV-2 ROD genomic RNA and mutations in the encapsidation domain. (A) The landmark sequences with known functions are indicated by boxes, with their names indicated above. TAR, polyA signal, PBS, ψ, SL1, SD, and gag represent the transactivation region, the poly(A) signal domain, the primer binding site, the encapsidation signal, stem-loop 1, the major splice donor site, and the 5′ end of the Gag protein coding region, respectively. The secondary structure of nucleotides 380 to 444 is represented, with gray boxes highlighting the 10-nt palindromic sequence pal and the 6-nt palindromic sequence in the loop of the SL1 element. The short thick line below nt 380 to 408 represents the encapsidation signal ψ characterized in cell culture (15). (B) Replication kinetics of wild-type (open squares), ψ-negative (open triangles), and pal-negative (closed circles) viruses in C8166 cells. DNA plasmids were transfected into COS-7 cells, and viruses were isolated from the supernatant. A standardized amount of viral particles (10 ng of p27 capsid, as determined by ELISA) was used to infect permissive C8166 cells. Aliquots of supernatants were then assayed for reverse transcriptase activity. (C) (Top) Sequences of the wild-type and scrambled pal 392-401 regions for the clones used in this experiment. SCR1 was designed to both destroy the palindrome and disrupt a proposed ψ-SL1 interaction (23, 24). SCR2 is the complementary sequence to SCR1. The nucleotides different from the wild-type sequence are represented with lowercase letters. (Bottom) Replication kinetics of wild-type (open squares), SCR1 (open triangles), and SCR2 (closed circles) pal viruses in C8166 cells. DNA plasmids were transfected into COS-7 cells, and viruses were isolated from the supernatant. A standardized amount of viral particles (10 ng of p27 capsid, as determined by ELISA) was used to infect permissive C8166 cells. Aliquots of supernatants were then assayed for p27 capsid by ELISA. (D) (Top) Sequences of the wild-type and mutated pal 392-401 regions for the clones used in this experiment. Weak and strong pal sequences were chosen from in vitro studies in which they were shown to form weaker and stronger RNA-RNA duplexes, respectively, than the wild-type sequence (36). (Bottom) Replication kinetics of wild-type (open squares), weak (open triangles), and strong (closed circles) pal viruses in C8166 cells. Viral replication was followed as described for panel C.

Infectivity and genomic RNA encapsidation are affected by alternative ψ(pal) mutations and restored through reversion-associated mutations. (A) Single-round infectivity assay with P4-CCR5 reporter cells of viruses produced by transfected COS-7 cells. The mutated viruses used in this experiment are shown in Fig. 2. The infectivity assay is described in Materials and Methods. The error bars represent the standard deviations for duplicate experiments. (B) (Top) RNase protection assay of wild-type (lanes 4), weak (lanes 5), and weak G394A/T397C mutant (lanes 6) pal viruses. The controls were the undigested probe (lane 1) and the digested probe used with in vitro RNA transcript 1-470 (lane 2) or 1-561 (lane 3). (Bottom) Diagram of the radioactive antisense probe used in this experiment and the expected protected fragments. (C) Encapsidation yields of different pal-mutated viruses. The amounts of unspliced genomic RNA in viruses isolated from the medium of transfected COS-7 cells were quantified using an RNase protection assay. The numbers were then normalized first to the amount of viruses in the medium, as determined by p27 capsid ELISA, and second to the level of encapsidation by wild-type viruses, set at 100%, to give the encapsidation relative to that of the wild type. The error bars represent the standard deviations for two or three experiments.

Two nucleotide changes that appeared during long-term culture are solely able to restore viral replication in alternative ψ(pal) viruses. (A) (Top) Sequences of the wild-type, weak, and weak G394A/T397C mutant pal 392-401 regions for the clones used in this experiment. The reversion-associated mutations are boxed, and nucleotides different from the wild-type sequence are shown with lowercase letters. (Bottom) Replication kinetics of wild-type (open squares), weak (open triangles), and weak G394A/T397C mutant (closed circles) pal viruses in C8166 cells. Viral replication was followed as described in the legend to Fig. 1B. (B) (Top) Sequences of the wild-type and mutated pal 392-401 regions for the clones used in this experiment. (Bottom) Replication kinetics of wild-type (open squares), strong (closed diamonds), strong G394A mutant (closed triangles), strong C400T mutant (open circles), and strong G394A/C400T mutant (closed circles) pal viruses in C8166 cells. Viral replication was followed as described in the legend to Fig. 1B.

Secondary structure models of extended SL1 and stem B structures described in this work. (A) The HIV-2 ROD isolate (GenBank accession no. M15390) SL1 structure is represented with another stem structure that base pairs with the 3′ end of the pal element with a 5′-GGAG-containing motif located immediately downstream of the short SL1 structure. We chose the names stem B and stems C1 and C2 to describe the extended HIV-2 SL1 in comparison with the structure of the HIV-1 SL1 element, as described by Laughrea and colleagues (26). (B) Mutations in the pal elements of strong pal viruses disrupt stem B (left structure). An alternative, more stable base pairing is possible by a one-nucleotide shift with a single mismatch in the middle (middle structure). The reversion-associated mutation C400T stabilizes the alternative stem B, and the G394A mutation restores a partial GGA motif (right structure). (C) Mutations in the pal elements of weak pal viruses decrease the stability of stem B (left structure). The reversion-associated mutation T397C or G442A stabilizes the mutated stem B by restoring Watson-Crick base pairing, while G394A reversion restores a partial GGA motif (right structure). The reversion-associated mutations are boxed, and nucleotides different from the wild-type sequence are shown with lowercase letters. (D) The consensus stem B structure from the analysis of 37 HIV-2 and SIV sequences from the HIV Sequence Compendium 2005 is indicated, with the proportions of alternative base pairs shown to the right (27). (E) Secondary structure model of HIV-1 SL1 from the HxB2 isolate (GenBank accession no. K03455). The stems are named according to reference 26.