Influence of sequence identity and unique breakpoints on the frequency of intersubtype HIV-1 recombination

Abstract

Background: HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population.

Results: Increased sequence identity between viruses or RNA templates resulted in increased recombination frequencies, with the exception of the 115-A virus or RNA template. Analyses of the recombination breakpoints and mechanistic studies revealed that the presence of a recombination hotspot in the C3/V4 env region, unique to 115-A as donor RNA, could account for the higher recombination frequencies with the 115-A virus/template. Single-cycle infections supported proportionally less recombination than the in vitro reverse transcription assay but both systems still had significantly higher recombination frequencies than observed in the multiple-cycle virus replication system. In the multiple cycle assay, increased replicative fitness of one HIV-1 over the other in a dual infection dramatically decreased recombination frequencies.

Conclusion: Sequence variation at specific sites between HIV-1 isolates can introduce unique recombination hotspots, which increase recombination frequencies and skew the general observation that decreased HIV-1 sequence identity reduces recombination rates. These findings also suggest that the majority of intra- or intersubtype A/D HIV-1 recombinants, generated with each round of infection, are not replication-competent and do not survive in the multiple-cycle system. Ability of one HIV-1 isolate to outgrow the other leads to reduced co-infections, heterozygous virus production, and recombination frequencies.

Figure 1

Prevalence of unique HIV-1 recombinant forms (or intersubtype HIV-1 recombinants). The location of subtypes (e.g. A, C, G, etc), circulating recombinant forms (CRFs), and unique recombinant forms (URFs) are mapped in sub-Saharan Africa and specifically, Central Africa in panel A. The number of humans infected with the dominant subtypes, CRFs, and URFs in the world or in Central Africa is graphed in panel B. The proportion of specific intersubtype recombinants (A/D, A/C, and others) responsible for URF infections in Central Africa has been reported (C) [20–23]. Panel D provides a neighbor-joining phylogenetic tree to describe the genetic relationship of the C1 to C4 env sequences of 115-A, 120-A, 89-D, 122-D, and 126-D to other reference HIV-1 sequences.

Figure 2

Schematic representation of intra- and intersubtype recombination systems. Single cycle tissue culture system (panel A) for recombination employed heterozygous VSV-pseudotyped env particles produced by transient co-transfection of two genomic and two helper plasmids in 293T cells. Following production from 293T cells, virus particles were used to transduce MT4 cells. PCR products cleaved with BamHI and SacII were then cloned into vectors for transfection into E. coli followed by screening of blue and white colonies. Calculations for the frequency of recombination are outlined in the Materials and Methods. Structure of the genomic plasmids and reverse transcription products are shown in panel A. The in vitro experimental system is outline in panel B and involves reverse transcription across a donor RNA template that shares a region of homology with an acceptor RNA template upstream of a genetic marker (lacZ') on the acceptor RNA or a truncated, non-functional portion of the malT gene from E.coli on the donor template. The donor RNA also contains at its 3' end an extension which is used to selectively prime reverse transcription after hybridization of a complementary oligonucleotide. Processive copying of the donor template will yield lac^- genotypes, while template switching during reverse transcription of the retroviral sequence will produce lac⁺ genotypes. The resulting double-stranded DNAs are restricted with BamHI and PstI and, after ligation to a plasmid vector, used for bacterial transformation. On appropriate media, recombinant DNAs will yield blue colonies distinguishable from the white colonies given by the parental DNAs. The same LacZ screening system is employed for single cycle assay (A). Multiple cycle tissue culture system (panel C) was performed by infecting U87.CD4.CXCR4 cells with subtype A and D HIV-1 isolates in pairs (0.001 MOI). After the first round of replication, co-infected cells can produce both parental and heterodiploid viruses. Infection of new cells with heterodiploid virions can lead to intersubtype recombination. The schema for PCR amplification of intersubtype HIV-1 env fragments is outlined in panel C and the calculation for frequency is described in the Materials and Methods. Finally, panel D describes the reconstituted in vitro reverse transcription assay which involves initiating HIV-1 DNA synthesis from a radiolabeled DNA primer annealed to a defined donor RNA template (e.g. C3-V4) and in the same reaction mixtures with an acceptor RNA template slightly longer and with a region of sequence homology with the donor to promote strand transfer. RNA-dependent DNA synthesis is catalyzed by reverse transcriptase and a 20 nt 5' [³²P]-labeled DNA primer on the RNA donor templates (225 nt), RNA acceptor templates (225 nt), and with or without NC. The templates have a 205 nt overlap region to promote intersubtype recombination. Products from these reactions were resolved on a 8% denaturing polyacrylamide gel.

Figure 3

Frequency of inter- and intrasubtype HIV-1 recombination in an in vitro, single cycle, and multiple cycle assay systems. Panel A indicates the nucleotide genetic distances that separate the env genes in each pair of subtype A and D primary HIV-1 isolates employed in this study. The recombination frequencies of each pair in panel B were calculated in three systems. For in vitro, the synthesis of minus strand DNA on the donor RNA template was catalyzed by RT. Products were PCR amplified, cloned, and blue/white colonies were screened to calculate recombination frequencies. For the single-cycle system, recombination occurred in a cell infected with a heterozygous virus particle. Recombinants were identified by PCR and by the same blue/white colony screening described in Figure 1 and in the Materials and Methods. Finally, the recombination frequency in the multiple cycle system was measured by quantitative PCR using isolate- or subtype-specific primers (see Materials and Methods and Figure 4). The sequence identity between each HIV-1 pair is shown as line graph with the scale on right of panel B. Panel C shows a plot of recombination frequency in the single cycle (filled circle) or in vitro (open circle) systems versus sequence identity. The r value represents the Pearson product moment correlation for in vitro (open circle) and single cycle (filled circle) assays.

Figure 4

Measuring fitness and recombination frequency in the multiple cycle system. U87.CD4.CXCR4 cell cultures dually infected with two isolates of different subtypes (A+D; panel A) or the same subtype infections (A+A or D+D; panel B) and then harvested for analyses. Subtype or isolate-specific primers were employed to amplify parental or recombinant HIV-1 env DNA (X axes) from specific dual infections (Z axes). Copy numbers on the Y axes were derived from control PCR amplifications with known copy numbers of subtype A and D DNA templates (10² to 10⁸ copies/reaction) (see Materials and Methods). Relative fitness values (W) and frequencies of recombination from these dual infections/competitions were calculated as described in the inset of panel C. Briefly, conserved primers were utilized to PCR amplify the env genes from parental and recombinant env progeny from each dual infection to measure fitness by HTA[54,62,63]. These PCR products were then denatured and annealed to a radiolabeled env probe, which was amplified from a subtype E HIV-1 env clone (E-pTH22. DNA heteroduplexes specific for the each parental isolate were resolved on a 6% nondenaturating polyacrylamide gel. A sample autoradiograph and calculations of relative fitness is defined in panel D. A plot of the fitness differences (W_D = W_{more fit}/W_{less fit}) or of percent recombinants (right Y axis) for each dual infection pair is shown in panel E.

Figure 5

Pausing patterns and hotspots of intersubtype recombination during reverse transcription on the 115-A and 120-A RNA donor template. Hotspots of recombination were mapped in the C1-C4 region as part of a previous study (A). RNA-dependent DNA synthesis is catalyzed by reverse transcriptase and a 20 nt 5' [³²P]-labeled DNA primer on the 115-A and 120-A RNA donor templates (225 nt). Reactions were in the presence or absence of D-89 acceptor (225 nt) and NC (Figure 1D). Reactions were stopped at 30 s, 1, 2, 4, 8, 16, 32, and 64 min and run on a 8% denaturing polyacrylamide gel. Products of these reactions are shown in the autoradiographs of panel B (A-115 donor) and panel C (A-120 donor). The positions of the primer (P), and full extended products derived from the donor template (D, 225 nt) and the strand transfer products (T, 245 nt). A major pause site during DNA synthesis was observed in the C3 region of 115-A donor template and is indicated by the "dumbbell" symbol. A putative V4 stem-loop is also outlined (see Figure 5 for details). Graphs of transfer efficiency vs. time for reactions with A-115 as both donor and acceptor (panel D) or A-115 (circles) or A-120 (triangles) as donor and D-89 as acceptor (panel E) are shown. Filled shapes are without NC and open with. The % transfer efficiency is defined as the amount of transfer product (T) divided by the sum or transfer plus full-length donor directed (D) products times 100 ((T/(T + D)) × 100).

Figure 6

Schematic of reverse transcription and template switching from the 115A to the 89D RNA templates. A 350 nt RNA sequence from position 7150 to 7500 (HXB2 numbering) of virus 115A, 120A, 89D, 122D, and 126D was submitted to Mfold server at MacFarlane-Burnett for RNA structure prediction based on the Zucher algorithms. The RNA structures for the five different templates varied considerably but one stem-loop or hairpin was consistently present around nt 7301 to 7339 and was termed the V4 stem-loop. Only 115A and 89D RNA structures between nt 7296 and 7421 are presented in this figure. Shifting the 350 nt RNA template 50 nt upstream or downstream did not affect the structure of this predicted V4 stem-loop. This RNA structure also contains the strong pause site identified in Figure 4B (blue dumbbell). The structure of virus 115A RNA was examined by sliding a 50 nt window from nt 7276 to 7126 on the 3' end and from nt 7235 to 7075 on the 5'end. To mimic the double stranded DNA/RNA duplex generated by (-) strand DNA synthesis by reverse transcriptase, a 20 nt duplex was added into the submitted sequence at the 3' end (designated as twenty "X's" in the sequence). The progression of RT on the 115-A RNA template for a 50, 100, and 150 nt extension is illustrated. The promiscuous template switching event to the 89-D template, near V4 stem-loop is also depicted. The "red" nucleotides represent the mismatched sequences between 115-A and 89-D templates. Recombination sites were identified and mapped to the sequences between these base mismatches. The "green stars" represent the recombination breakpoints identified in 16 recombinant clones from the reconstituted in vitro assay in the absence of NC (Figure 4B) and "pink squares", those in 18 recombinant clones from the reconstituted in vitro assay in the presence of NC (Figure 4B). The "blue circles" represent the breakpoints from five clones of the single cycle infection assay, i.e. identified in the C3-V4 region).