Infectious molecular clone of a recently transmitted pediatric human immunodeficiency virus clade C isolate from Africa: evidence of intraclade recombination

Abstract

Although human immunodeficiency virus type 1 (HIV-1) clade C continues to dominate the pandemic, only two infectious clade C proviral DNA clones have been described (N. Mochizuki, N. Otsuka, K. Matsuo, T. Shiino, A. Kojima, T. Kurata, K. Sakai, N. Yamamoto, S. Isomura, T. N. Dhole, Y. Takebe, M. Matsuda, and M. Tatsumi, AIDS Res. Hum. Retrovir. 15:1321-1324, 1999; T. Ndung'u, B. Renjifo, and M. Essex, J. Virol. 75:4964-4972, 2001). We have generated an infectious molecular clone of a pediatric clade C strain, HIV1084i, which was isolated from a Zambian infant infected either intrapartum or through breastfeeding. HIV1084i is an R5, non-syncytium-inducing isolate that bears all known clade C signatures; gag, pol, and env consistently mapped within clade C. Interestingly, gag resembled Asian isolates, whereas pol and env resembled African isolates, indicating that HIV1084i probably arose from an intraclade recombination. As a recently transmitted clade C strain, HIV1084i will be a useful vaccine development tool.

FIG. 1.

Strategy for the cloning of HIV1084i. Full-length HIV1084i was constructed from two subgenomic amplicons containing NotI and AscI restriction sites at alternate ends of the molecule. NotI restriction sites were added to the LTR primers (1 and 4), while AscI restriction sites were introduced into primers 2 and 3, which spanned the vpr open reading frame. Subcloning the PCR product into pCR 2.1 Topo cloning vectors, followed by bacterial amplification, restriction endonuclease-mediated linearization, and subsequent ligation yielded the 14.7-kb proviral plasmid, HIV1084i.

FIG. 2.

Kinetics of replication of HIV1084i and Indie-C1 in PBMCs with or without AZT. PBMCs with or without 10 μM AZT were infected with excess HIV1084i or HIV Indie-C1. Supernatants were collected at various days postinfection and analyzed by p24 Gag ELISA. The figure depicts the average of results from two independent experiments.

FIG. 3.

Phylogenetic analysis of gag, pol, env, Vpu, and Rev. Using Clustal X (version 1.81) followed by PAUP (version 4.0), unrooted bootstrapped phylogenetic trees were generated for gag (A), pol (B), env (C), Vpu (D), and Rev (E) of HIV1084i. One thousand bootstrap replicates, a gap opening penalty of 50 (or 10), a gap extension penalty of 5 (or 0.1), and the International Union of Biochemistry DNA (or Gonnet 250 protein) weight matrix were used to generate the trees. Only bootstrap values greater than 70 are indicated. All reference DNA sequences were obtained from the Los Alamos National Laboratory HIV database (http://hiv-web.lanl.gov/). 93IN101 (AB023804) is referred to herein as Indie-C1 (9).