AEGiS-15IAC: Isolation and biological characterization of a replication-competent molecular clone of human immunodeficiency virus type 1 circulating recombinant form 08

AEGiS-15IAC: Isolation and biological characterization of a replication-competent molecular clone of human immunodeficiency virus type 1 circulating recombinant form 08_BC.

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

Isolation and biological characterization of a replication-competent molecular clone of human immunodeficiency virus type 1 circulating recombinant form 08_BC.

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. TuOrA1142)

Takebe Y, Kusagawa S, Yang R
National Institute of Infectious Diseases, Tokyo, Japan

BACKGROUND: The infectious HIV-1 molecular clones have been critical tools for systemic evaluation of the structure and function of HIV-1 genes and for delineating the mechanisms of viral replication and pathogenesis. In the present study, we isolated and characterized the infectious molecular clones for CRF08_BC that caused the explosive epidemic among injecting drug users (IDUs) in southeastern China.

METHODS: A CRF08_BC strain (00CN-HH040) was isolated from an IDU in Yunnan Province of China. The infectious molecular clone was reconstituted by PCR-based amplification-cloning method, involving the direct ligation of 8.3-kb proviral amplicons into a recovery vector carrying two functional LTRs. The infectivities to peripheral blood nuclear cells (PBMCs) and primary macrophages and coreceptor usages were examined. The complete HIV-1 nucleotide sequence was determined and subjected to the recombination breakpoint analyses to verify the subtype structure.

RESULTS: We successfully reconstituted an infectious molecular clone of CRF08_BC (designated 00CN-HH040. NX22) from a Yunnan HIV-1 isolate. 00CN-HH040. NX22 replicated to high titers in PBMCs. This clone utilized only CCR5 as a coreceptor for entry, but did not replicate in primary macrophages. The recombination breakpoint analyses demonstrated that 00CN-HH040. NX22 shared identical structural profile to that of CRF08_BC reference strain. The sequence signatures unique to CRF08_BC were identified in the enhancer region of LTR.

CONCLUSIONS: We isolated and biologically characterized an infectious molecular clone for CRF08_BC. This represents the first report of the isolation of an infectious molecular clone for CRF08_BC. This clone will be useful tool to delineate the virological properties of CRF08_BC and may also be used in design for vaccine candidates to limit the epidemic in China.

Keywords: AEGIS, HIV-1, Virus Replication, Receptors, CCR5, Recombination, Genetic, Clone Cells, Blood Transfusion, Polymerase Chain Reaction, Macrophages, China, Humans, virology, genetics

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TuOrA1142