Figure 1.
Construction of HIV-1 CRF08_BC infectious clone.
Proviral DNA from 2007CNGX-HK, without its homologous LTRs, was PCR-amplified and subcloned into pNL4-3. This resulted in the chimeric clone pNGX5 harboring a majority of the genome of 2007CNGX-HK, that is, except for the homologous LTRs as indicated. Subsequently, the full-length genome of pNGX5 was subcloned in pBR322 after AatII and NgoMIV digestion. The 5′ LTR and 3′ LTR of the resultant plasmid pBRNG were then exchanged with those from 2007CNGX-HK, using AatII and SpeI restriction digestion for the 5′ LTR and using NgoMIV and XhoI restriction digestion for the 3′ LTR. The final construct pBRGX was 12,701 bp and was replication-competent.
Figure 2.
Replication kinetics of HIV-1 CRF_08BC molecular clones pBRNG and pBRGX and its parental isolate.
(A) The p24 levels in the supernatants from pBRNG and pBRGX-transfected 293FT cells are shown. (B) The p24 levels in the PBMCs infected with pBRNG and pBRGX-derived virus, as well as the parental isolate are noted at days 3, 7, 10, 13 and 17 post-infection. An ELISA assay was used to assay the p24 antigen concentrations.
Figure 3.
Alignment of the LTR sequences from pNL4-3 and 2007CNGX-HK.
Both LTRs from pNL4-3 and 2007CNGX-HK were sequenced and their lengths were noted to be similar, at 634 bp and 632 bp respectively. However, there were seventy-eight base substitutions, six base deletions and four insertions observed between these two LTRs. There is a potential third NF-κB binding site (NF-κB iii) in the parental (2007CNGX-HK) LTR region due to the four base insertions at nt 340–343. Three single base deletions are also found amongst the NF-κB i, SP-1 iii and SP-1 i binding sites, respectively. The three major regulatory regions of LTR are indicated by vertical bars: Modulatory (NF-AT, USF, TCF1α, NF-κB), Core (SP-1, TATA box) and TAR (trans-activation region) elements. The green underlined sequences indicate selected base substitutions, insertions and deletions. Sequence identity is indicated by dots; insertions/deletions are indicated by dashes.
Figure 4.
Co-receptor-mediated tropism of the pBRGX construct and its parental primary isolate.
U373-MAGI-CXCR4cem (CXCR4), U373-MAGI-CCR5e (CCR5) and U373MAGI (none) cells were infected with either pBRGX-derived virus or its parental primary isolate. The infectivity of the virus was tested by the X-Gal Staining assay. The number of positives were scored per well and indicated along the y-axis.
Table 1.
Base substitution mutations in the HIV-1 infectious clone pBRGX coding proteins as compared with the parental primary isolate 2007CNGX-HK.
Figure 5.
Similarity plots for the pBRGX strain.
Genome recombination analysis of the pBRGX strain versus the reference subtype strains obtained from the Los Alamos database (http://www.hiv.lanl.gov/content/index), including Consensus_A1, B, C, D, F1, F2, G, H, 01AE and CRF08_BC.97CNGX_6F, are presented. SimPlot analysis was performed with web-accessible software, RIP 3.0 (http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html), using a window size of 400 bp and a step size of 50 bp. As per the software protocol, the alignment was gap-stripped before analysis.
Figure 6.
Schematic representation of the recombinant clone of pBRGX.
The map of the construct pBRGX was generated using the Recombinant HIV-1 Drawing Tool, available from the LANL site (http://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html). Subtype B′ sequences are shown in red and subtype C sequences are displayed in white. The positions of six breakpoints are expressed as HXB2 coordinates at nt 1322, nt 1563, nt 2824, nt 3138, nt 8881 and nt 8950, respectively.
Figure 7.
Phylogenetic reconstruction of HIV-1 subtypes and recombinants of Asian origin.
A phylogenetic tree was generated from sequence alignments, and plotted using a neighbor-joining method. This was analysis implemented in MEGA 5 [35], [36] using 1000 bootstrap replicates. The optimum tree, with the sum of branch length equal to 2.05356961 is displayed. The evolutionary distances were computed using the Maximum Composite Likelihood method, available as a public software tool (http://www.megasoftware.net/WebHelp/part_iv___evolutionary_analysis/computing_evolutionary_distances/distance_models/nucleotide_substitution_models/hc_mcl.htm). The analysis included 113 nucleotide sequences in toto. Reference HIV-1 genomic sequences of subtypes B, C, 07BC and 08BC were used, and are available from the Los Alamos National Library (LANL). All positions containing gaps and missing data were eliminated (gap-stripped) as per the software algorithm. The 2007CNGX-HK genome formed a clade with the other CRF_08BC isolates. The grey circles indicate the junctions of the branches having close phylogenetic relationships.