Abstract
BackgroundThe pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. This situation is more complex in subtype C infection where coreceptor switch is either absent or extremely rare. To gain insights into the mechanisms that underlie coreceptor requirement of subtype C, we screened several primary viral isolates and identified a clinical sample that demonstrated a potential to grow on standard T-cell lines with no detectable CCR5 expression. The subject was diagnosed with HIV-1 associated dementia in the absence of opportunistic infections of the brain. To isolate molecular clones from this virus, we devised a novel strategy based on anchor primers that target a sequence in the reverse transcriptase, highly conserved among diverse subtypes of HIV-1.ResultsUsing this strategy, we isolated 8 full-length molecular clones from the donor. Two of the eight molecular clones, 03In94_D17 and 03In94_D24, (D17 and D24) generated replication-competent viruses. Phylogenetic analysis of the full-length viral sequences revealed that both clones were non-recombinant subtype C viruses. They contain intact open reading frames in all the viral proteins. Both the viral clones are endowed with several unique molecular and biological properties. The viral promoter of the clones is characterized by the presence of four NF-kB binding elements, a feature rarely seen in the subtype C HIV-1 LTR. Interestingly, we identified the coexistence of two different forms of Rev, a truncated form common to subtype C and a full-length form less common for this subtype, in both proviral and plasma virus compartments. An exceptional property of the viruses, atypical of subtype C, is their ability to use a wide range of coreceptors including CCR5, CXCR4, and several others tested. Sequence analysis of Env of D17 and D24 clones identified differences within the variable loops providing important clues for the expanded coreceptor use. The V1, V2 and V4 loops in both of the molecular clones are longer due to the insertion of several amino acid residues that generated potential N-linked glycosylation sites.ConclusionThe exceptional biological and molecular properties of these clones make them invaluable tools to understand the unique pathogenic characteristics of subtype C.
Highlights
The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood
Isolation of molecular clones from an Indian donor with HIV-associated dementia (HAD) Virus isolated from the plasma of an Indian donor with HAD (BL94/03) proliferated on HOS-CCR5, HOS-CXCR4 cells and certain T-cell lines that lacked detectable surface expression of CCR5 suggesting expanded coreceptor use
The region of reverse transcriptase (RT) selected for anchor primer binding and MluI engineering is highly conserved among all the major viral subtypes of HIV-1 (Figure 1, inset) viral genetic heterogeneity is not likely to influence the cloning efficiency
Summary
The pathogenic significance of coreceptor switch in the viral infection of HIV-1 is not completely understood. New viral infections are initiated by viral strains that require the coreceptor CCR5 on the target cell (R5 strains), regardless of the subtype nature [10,11]. Towards the later stages of the infection, nearly half of subtype B strains switch their coreceptor usage to CXCR4 (X4 strains) [12,13]. Coreceptor switch from CCR5 to CXCR4 is less common in subtype C infection [8,9,19,20,21,22], the pathological significance of which, is not understood. The actual incidence of CXCR4 using subtype C strains in natural infection, the time of their appearance in the disease progression and the relevance of their emergence have not been elucidated
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