Infectious molecular clones with the nonhomologous dimer initiation sequences found in different subtypes of human immunodeficiency virus type 1 can recombine and initiate a spreading infection in vitro

Abstract

Recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been shown to be of major importance in the global AIDS pandemic. Viral RNA dimer formation mediated by the dimerization initiation sequence (DIS) is believed to be essential for viral genomic RNA packaging and therefore for RNA recombination. Here, we demonstrate that HIV-1 recombination and replication are not restricted by variant DIS loop sequences. Three DIS loop forms found among HIV-1 isolates, DIS (CG), DIS (TA), and DIS (TG), when introduced into deletion mutants of HIV-1 recombined efficiently, and the progeny virions replicated with comparable kinetics. A fourth DIS loop form, containing an artificial AAAAAA sequence disrupting the putative DIS loop-loop interactions [DIS (A6)], supported efficient recombination with DIS loop variants; however, DIS (A6) progeny virions exhibited a modest replication disadvantage in mixed cultures. Our studies indicate that the nonhomologous DIS sequences found in different HIV-1 subtypes are not a primary obstacle to intersubtype recombination.

FIG. 1

Predicted DIS RNA stem-loop structures of NL4-3 and NL4-3 DIS variants. The DIS mutants were generated by site-directed mutagenesis; for details, see Materials and Methods. The MFold program predicts the hairpin structures of all of the DIS variants; thus, alteration of the loop sequence minimally impacts stem structure. The palindromic motifs in the loop are in boldface, and restriction enzymes with corresponding cleavage sites (boxed) are shown above the hairpin loop. Variations at the second and fifth positions of the palindrome occurring in HIV-1 subtypes are marked (*). Phylogenetic association of DIS RNA elements of different HIV-1 strains is given in Table 1.

FIG. 2

Replication kinetics of NL4-3 containing variant DIS. SupT1 cells were infected with NL4-3 DIS (TA) (squares), NL4-3 DIS (CG) (circles), and NL4-3 DIS (TG) (triangles) at an MOI of 0.001, and virus-associated p24^gag antigen production was determined at designated time points. The inset shows provirus DIS PCR products at day 12 in culture cleaved with restriction endonucleases capable of distinguishing DIS palindromic sequence. DIS PCR product restriction enzyme digestion results in the production of 89- and 76-bp DNA fragments.

FIG. 3

(A) Recombination between heterologous RNA during replication results in four proviral forms, the two parental forms, NL4-3Δpol and NL4-3Δenv, and two recombinant forms, wild-type NL4-3 (NL4-3WT) and NL4-3Δpol/Δenv. Arrows denote oligonucleotides used for PCR amplification (Rec1, Rec2, DIS1, and DIS2) and hybridization (Std*, Pol*, and ENV*) and the direction of sequence complementarity. Oligonucleotides marked with asterisks are probes for detecting PCR products. Hatch marks denote unrepresented sequences in HIV-1. Long terminal repeat (LTR) and structural genes are shown. (B) Provirus resulting from a single-cycle infection of SupT1 cells. Wild-type NL4-3 DIS (CG) and virus generated by cotransfection of NL4-3Δpol DIS (CG) and NL4-3Δenv DIS (CG) were used to infect SupT1 cells. Lysates of infected cells were amplified with primers Rec1 and Rec2, and proviral sequences were identified by hybridization with Std*, Pol*, and ENV* probes. Molecular weights of the PCR products confirm expected recombination products. (C) DNA recombination during virus production. Hirt supernatants from 293 cells transfected with no DNA, pNL4-3Δenv DIS (CG), pNL4-3Δenv DIS (CG) and NL4-3Δpol DIS (CG), NL4-3Δpol DIS (CG), or NL4-3 DIS (CG) were amplified with primers Rec1 and Rec2, and proviral sequences were identified by hybridization with the Std* probe. Lysate of SupT1 cells infected (Inf) with virus generated by cotransfection of NL4-3Δpol DIS (CG) and NL4-3Δenv DIS (CG) was amplified as a control. WT, wild type.

FIG. 4

(A) Recombinant forms of provirus observed in single-cycle infection of SupT1 cells. SupT1 cells were infected with various viral stocks at an MOI of 0.001. Provirus formation was analyzed by PCR, and recombinant viral PCR products were identified by hybridization to Std* probe 24 h after infection (Fig. 3B). PCR products derived from wild-type (WT), parental, and double-deleted mutant provirus are indicated. Provirus bands hybridizing with the probe were quantitated with a PhosphorImager, and the efficiency of recombination was determined by comparing the amount of wild-type and double-deleted provirus formed to the total amount of all provirus structures observed. (B) Replication kinetics of HIV-1 resulting from recombination of NL4-3 DIS variants. SupT1 cells were infected at an MOI of 0.0001 with equivalent MAGI cell infectious units of NL4-3 DIS (TA) and replication-competent recombinant virus resulting from recombination between NL4-3Δpol and NL4-3Δenv DIS variants. Virus-associated p24^gag antigen production was measured at the designated time points. Left, kinetics of replication of recombinant virus resulting from recombination between NL4-3Δpol DIS (TA) and NL4-3Δenv DIS (TA) (squares), NL4-3Δenv DIS (CG) (triangles), and NL4-3Δenv DIS (TG) (open circles). Replication kinetics of wild-type NL4-3 DIS (TA) is also shown (diamonds). Center, kinetics of replication of recombinant virus resulting from recombination between NL4-3Δpol DIS (CG) and NL4-3Δenv DIS (TA) (squares), NL4-3Δenv DIS (CG) (triangles), and NL4-3Δenv DIS (TG) (circles). Right, kinetics of replication of recombinant virus resulting from recombination between NL4-3Δpol DIS (TG) and NL4-3Δenv DIS (TA) (squares), NL4-3Δenv DIS (CG) (triangles), and NL4-3Δenv DIS (TG) (circles). (C) DIS element identification in virus recombinants during culture. The DIS loop sequence of replication-competent recombinant provirus was analyzed in cell lysates at day 14 in culture by DIS PCR followed by restriction enzyme digestion of the PCR product (Fig. 3) as follows: B, BssHII; A, ApaLI; F, FspI; U, uncut. Complete restriction enzyme digestion of the DIS PCR product is observed in cultures containing recombinant provirus resulting from recombination of homologous DIS. Partial digestion of DIS PCR product is observed in recombinant provirus resulting from recombination of heterologous DIS. Identical results observed at earlier time points (days 7 and 10) are not shown.

FIG. 5

Replication kinetics of NL4-3 DIS (A6). SupT1 cells were infected at an MOI of 0.001 with NL4-3 DIS (A6) (diamonds) and recombinant virus resulting from recombination between NL4-3Δenv DIS (A6) and NL4-3Δpol DIS (TA) (squares), NL4-3Δpol DIS (CG) (triangles), and NL4-3Δpol DIS (TG) (circles), and virus-associated p24^gag antigen production was determined at designated time points. Dynamics of recombinant virus populations in culture are shown in Table 3.