Relative expression of <i>rev</i> RNAs in subtype C viruses according to peak areas in GeneMapper analyses.

<p>Results correspond to peaks shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone-0030574-g002" target="_blank">Fig. 2</a>, and are shown as % of individual peak areas relative to the sum of peak areas of all <i>rev</i> RNA-derived products. Percentages at the column on the right correspond to the cloned and sequenced <i>rev</i> RNA-derived amplicons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone-0030574-t001" target="_blank">Table 1</a>).</p>1<p>A small 331 nt peak, coincident with that of 1.4g.7 <i>rev</i> RNA, was seen in X1702-3 and X1936 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone-0030574-g002" target="_blank">Fig. 2</a>). However, nested PCR using an antisense primer specific for <i>rev</i>, <i>tat</i> and <i>vpr</i> RNAs failed to detect 1.4g.7 <i>rev</i> RNA in these isolates.</p>2<p>Nested PCR with primers recognizing exons 2 and 3 allowed to confirm that these products, only 1 nt longer in X1702-3 and X1936 than in X2363-2, correspond to 1.3.4f.7 in the first two viruses and to 1.2.4g.7 in the third one.</p>3<p>The 367 nt peak seen in X2363-2 may correspond to both 1.2.4c.7 and 1.3.4b.7 <i>rev</i> RNAs. Nested PCR using primers recognizing exons 2 and 3 allowed to determine that this peak corresponds to 1.2.4c.7.</p

GeneMapper analyses of DS RNAs expressed by three HIV-1 subtype C primary isolates in PBMCs.

<p>Green peaks represent PCR products and orange peaks represent size standards. Size of PCR product, encoded gene, and exon composition (named as in previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Schwartz1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Purcell1" target="_blank">[2]</a>) predicted according to the size of the PCR product are shown on top or on the side of each peak. Peaks whose sizes do not match HIV-1 transcripts using previously reported splice sites are marked with interrogation signs. For each subtype C virus, three GeneMapper analyses are shown, corresponding to infections using PBMCs from three different donors.</p

Schematic representation of HIV-1 splicing.

<p>Open reading frames are shown as open boxes and exons as black bars. Exons are named as previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Schwartz1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Purcell1" target="_blank">[2]</a>. All spliced transcripts incorporate exon 1. Optionally, noncoding exons 2 or 3 or both can be incorporated into <i>tat</i>, <i>rev</i>, <i>nef</i>, or <i>env</i> transcripts, and exon 2 into <i>vpr</i> transcripts. Proteins encoded in spliced RNAs are indicated on the right of the 3′ exon.</p

Exon composition of clones derived from DS <i>rev</i> and <i>tat</i> RNAs expressed by three subtype C isolates*.

<p>Exons 1, 2, 3, 4c, and 4 are named as in previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Schwartz1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Purcell1" target="_blank">[2]</a>. Exons 4f and 4g designate those using newly identified 3'ss A4f and A4g, respectively, and exon 2a designates that using 3'ss A1 and newly identified 5'ss D2a (see main text). All transcripts are assumed to include exon 7 through splicing from 5'ss D4 to 3'ss A7. However this splice junction was not sequenced, since the antisense PCR primer anneals upstream of D4. <i>nef</i> RNAs were not amplified because the antisense PCR primer recognizes a sequence upstream of 3'ss A5.</p

Intronic and exonic sequences surrounding newly identified splice sites in three subtype C isolates.

<p>Sequences are aligned with consensuses of subtypes B and C. (a) Sequences surrounding 3'ss A4f and A4g. AG dinucleotides in the intron ends adjacent to splice sites are in bold type. Polypyrimidine tracts potentially used for splicing at A4f and A4g are boxed. The sequences of subtype B NL4-3 and SF2 isolates are on bottom with branch sites previously identified for <i>rev</i> RNA splicing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Bilodeau1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Swanson1" target="_blank">[14]</a> underlined. Nucleotides in the subtype C isolates and in the consensus subtype C sequence potentially used as branch points for splicing at A4f and A4g (see main text) are indicated with arrows. (b) Sequences surrounding 5'ss D2 and D2a. Exon-intron borders are signaled with vertical lines. Highly conserved GU dinucleotides at intron ends adjacent to the 5'ss are in bold type. Nucleotides at splice sites potentially pairing with U1 snRNA are underlined.</p

Sequence electropherograms of splice junctions newly identified in subtype C isolates.

<p>Splice junctions are shown as vertical lines. 5′ and 3′ splice sites involved in splicing, named as in previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Schwartz1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030574#pone.0030574-Purcell1" target="_blank">[2]</a> and in this study (see main text), are signaled, with nucleotide positions in the HXB2 genome in parentheses. Nearby splice sites are also indicated.</p

ML trees of sequences of HIV-1 isolates from databases (all from Portugal) or obtained by us (from Spain) clustering with the BG recombinant virus 9196_01 in (a) PR-RT and/or the (b) V3 region.

<p>Sequences obtained in this study are in bold type. HXB2 positions delimiting the analyzed segments are in parentheses. Countries of collection of database viruses of subtype G in PR-RT and of subtype B in V3 are indicated with the two-letter ISO code. Only bootstrap values ≥70% are shown.</p

Analysis of the relationship of CRF73_BG with CRF14_BG.

<p>(a) ML tree of concatenated subtype G fragments of X3208 and 9196_01, analyzed with G_Ib and CRF14_BG viruses. Countries of collection of subtype G viruses are indicated with the two-letter ISO code. (b) ML tree of the 5’ <i>env</i> fragment. (c) ML tree of the subtype B 3’ <i>env</i> fragment. HXB2 positions delimiting the analyzed segments in (b) and (c) are in parentheses. CRF73_BG viruses are in bold type. Countries of collection of database viruses of subtype G viruses in (a) and of subtype B viruses in (b) and (c) are indicated with the two-letter ISO code. Only bootstrap values ≥70% are shown.</p

Intersubtype breakpoint locations in HIV-1 BG recombinant viruses analyzed in this study, including all available CRF14_BG viruses sequenced in near full-length genomes, as determined with jpHMM.

<p>Intersubtype breakpoint locations in HIV-1 BG recombinant viruses analyzed in this study, including all available CRF14_BG viruses sequenced in near full-length genomes, as determined with jpHMM.</p

Identification of an HIV-1 BG Intersubtype Recombinant Form (CRF73_BG), Partially Related to CRF14_BG, Which Is Circulating in Portugal and Spain

<div><p>HIV-1 exhibits a characteristically high genetic diversity, with the M group, responsible for the pandemic, being classified into nine subtypes, 72 circulating recombinant forms (CRFs) and numerous unique recombinant forms (URFs). Here we characterize the near full-length genome sequence of an HIV-1 BG intersubtype recombinant virus (X3208) collected in Galicia (Northwest Spain) which exhibits a mosaic structure coincident with that of a previously characterized BG recombinant virus (9601_01), collected in Germany and epidemiologically linked to Portugal, and different from currently defined CRFs. Similar recombination patterns were found in partial genome sequences from three other BG recombinant viruses, one newly derived, from a virus collected in Spain, and two retrieved from databases, collected in France and Portugal, respectively. Breakpoint coincidence and clustering in phylogenetic trees of these epidemiologically-unlinked viruses allow to define a new HIV-1 CRF (CRF73_BG). CRF73_BG shares one breakpoint in the envelope with CRF14_BG, which circulates in Portugal and Spain, and groups with it in a subtype B envelope fragment, but the greatest part of its genome does not appear to derive from CRF14_BG, although both CRFs share as parental strain the subtype G variant circulating in the Iberian Peninsula. Phylogenetic clustering of partial <i>pol</i> and <i>env</i> segments from viruses collected in Portugal and Spain with X3208 and 9691_01 indicates that CRF73_BG is circulating in both countries, with proportions of around 2–3% Portuguese database HIV-1 isolates clustering with CRF73_BG. The fact that an HIV-1 recombinant virus characterized ten years ago as a URF has been shown to represent a CRF suggests that the number of HIV-1 CRFs may be much greater than currently known.</p></div