Isolation and Characterization of a Replication-Competent Molecular Clone of an HIV-1 Circulating Recombinant Form (CRF33_01B)
Figure 1
Outline for constructing a replication-competent DNA clone of HIV-1 CRF33_01B (p05MYKL045.1).
Near full-length proviral DNA of CRF33_01B was amplified by long-range PCR using pbsA-NarI and 9KU5B primers and TA-cloned into a pCR-XL-TOPO vector. DNA clone containing CRF33_01B genome and p93JP-NH1, an infectious clone of CRF01_AE origin [13], were linearized by NarI and EcoRI. The NarI-EcoRI fragment from the respective CRF33_01B proviral DNA was directionally ligated with the pBR-SK2 vector that contains the p93JP-NH1 5′ long terminal repeat (LTR) to reconstitute a chimeric full-length construct. Each clone was purified and transformed into HeLa cells to determine proviral replication. Constructs producing non-replicating viruses were then rescued by reconstituting a 3.7 kb proviral fragment (with NcoI and EcoRI sites) that includes the functional env gene to recover an infectious CRF33_01B clone, designated as p05MYKL045.1. Restriction enzyme sites in the DNA and the p93JP-NH1-derived 5′ LTR region in p05MYKL045.1 (shaded) are indicated. Refer text for complete descriptions.