An Infectious DNA Clone of HIV Type 1 Subtype C

Abstract

Among the 10 subtypes of the M group of human immunodeficiency virus type 1, subtype C is the most prevalent in India and may dominate worldwide in the near future; however, there has been no report on the infectious DNA clone of this subtype. We have isolated an infectious DNA clone of the 93IN101 strain of HIV-1 subtype C, which was isolated in India in 1993. MAGIC5 cells, which are derived from HeLa-CD4-LTR-beta-gal (MAGI) cells and express CCR5, were inoculated with the 93IN101 strain of HIV-1 subtype C. The genomic DNA of the infected cells was used as a template for amplification of the HIV-1 genome. The genome DNA obtained was subcloned into pBR322, and the resulting plasmid was designated as pIndie-C1. The insert of pIndie-C1 was 9680 bp in length and had an intact genomic organization with open reading frames of all structural, regulatory, and accessory proteins. Phylogenetic analysis confirmed that the nucleotide sequence of pIndie-C1 is closely related to those of HIV-1 subtype C isolated in India. Transfection of pIndie-C1 into 293T cells yielded as much virus as did pNL432, one of the most widely used HIV DNA clones. The recovered Indie-C1 virus infected MAGIC5 but not the parent MAGI cells, indicating that Indie-C1 is CCR5 tropic. Expressed Env protein was reacted efficiently with the sera of HIV-1-infected patients of India, but not of Japan. Expression of Nef and Vpr was also confirmed by immunoblotting.

Human Immunodeficiency Virus Type 1 Subtype C Exhibits Higher Transactivation Activity of Tat than Subtypes B and E

“…In contrast, subtype C Tat proteins, especially in isolates from India, have serine instead of cysteine at amino acid residue 31 (15,32,38,49). In our study, the Tat activity of subtypes B and E Tat in the mouse NIH3T3 cells was affected by the substitution of amino acid Cys 31 to Ser 31 .…”

“…For the construction of the eukaryotic expression plasmids coding for the wild-type Tat (thereafter named pTat) derived from the three subtypes, the cDNAs including the tat gene were synthesized from the total RNA extracted from 293T cells transfected with the infectious HIV-1 clones, pNL4-3 in subtype B (2), p95TNIH022 in subtype E (43), and pIndie-C in subtype C (38), by reverse transcription (RT) using oligo-dT primers. Polymerase chain reaction (PCR) amplifications for full-length tat genes were carried out using the primers listed in Table 1: T1 for B-Tat and CTat and T2 for E-Tat as forward primers and T3 for BTat, T4 for C-Tat, and T5 for E-Tat as reverse primers.…”

Detection of IgA‐Binding Sites on Human Immunodeficiency Virus Type‐1 Envelope Glycoproteins, Gp120 and Gp41

“…In some experiments, IgA antibodies in plasma were precipitated using agarose con- for 1 hr at 37 C. After washing with PBS, the cells were cultured at 37 C in 5% CO,. The cytopathic effects of U87-CD4-CXCR4 cells were examined at day 6 (11) and the infected MAGIC-5A cells were counted at day 5 as previously described (14).…”

Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV

“…The serially diluted antisera were incubated with 200-300 blue spot-forming units (BFU) of HIV-1 LAI at 371C for 1 h. The mixture was incubated with confluent MAGIC5 cells (from Dr Tatsumi, National Institute of Infectious Diseases, Tokyo, Japan), 50,51 Dulbecco's modified Eagle's medium (DMEM) with 10% FCS and 0.2 mg/ml of G418 in a 96-well plate at 371C for 2 days. The cells were fixed with fixing solution (1% formaldehyde, 0.2% glutaraldehyde in PBS) for 5 min and stained with staining solution (4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM magnesium chloride, 0.4 mg/ml X-gal in PBS) at 371C for 18-24 h. The staining was stopped by removing the staining solution and the cells were washed twice with PBS.…”

“…The blue spot in each well was counted after the staining, and the neutralizing titer was calculated as (1À(% infection/% infection of control wells)) Â 100. The 50% neutralization dose (ND 50 ) is defined as the concentration of the Ab that reduced the number of infected cells by 50%. The detecting limitation of the assay was 100 ND 50 /ml.…”