UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG

HIV Pathogenesis Research Laboratory

Mission

To increase the knowledge base of HIV-1 subtype C pathogenesis through basic research that is directly applicable to novel drug development and vaccine design.

Activities:

Teaching

Dr Papathanasopoulos and Dr Capovilla are each actively involved in teaching several topics of the Honours course, Molecular Medicine II curriculum, as well as the Haemotology block of GEMP I. In addition Dr Capovilla lectures the Haematology registrars. They both have postgraduate students that they supervise (see above).

Research

Publications

  1. Wallis C.L., Rekhviashvili N., Papathanasopoulos M.A., Stevens G. and Stevens W.S. A Sensitive Real-time HIV-1 Drug Resistance Assay for the identification of the K65R mutation associated with cross-resistance to several nucleoside reverse transcriptase inhibitors. (submitted).

  2. Bell C.M., Connell B.J., Capovilla A., Venter W.D.F., Stevens W.S. and Papathanasopoulos M.A. (2007) Molecular characterization of the HIV-1 subtype C accessory genes vif, vpr and vpu. AIDS Research and Human Retroviruses. (In press).

  3. Coetzer M., Cilliers T., Papathanasopoulos M., Ramjee G., Abdool Karim S., Williamson C. and Morris L (2007). Longitudinal analysis of HIV-1 subtype C envelope sequences from South African patients. AIDS Research and Human Retroviruses. (In press).

  4. Brandful J.A.M., Coetzer M.E., Cilliers T., Phoswa M., Papathanasopoulos M.A., Morris L. and Moore P.L. (2007). Phenotypic characterization of HIV-1 isolates from Ghana. AIDS Research and Human Retroviruses. (In press).

  5. Choge I., Cilliers T., Walker P., Taylor N., Phoswa M., Meyers M., Viljoen J., Violari A., Gray G., Moore P., Papathanosopoulos M. and Morris L. (2006). Genotypic and phenotypic characterization of viral isolates from HIV-1 subtype C infected children with slow and rapid disease progression. AIDS Research and Human Retroviruses. 22:458-465.

Research and development activities

Virology: Ongoing Projects

1. Reagents and Assays to monitor HIV-1 subtype C antiviral drug susceptibility and/or resistance.

a. Generation of well characterized HIV-1 subtype C viral stocks

We have established a well characterized repository of HIV-1 subtype C primary isolates from AIDS patients and patients failing therapy (ARV drug resistant viruses). All the isolates have been genotypically and phenotypically characterized. A panel of CCR5 and CXCR4-utilizing viruses has been selected and utilized for the in vitro phenotypic sensitivity assays.

b. CD4-independent isolates (Bridgette Connell)

We have successfully adapted isolate 05ZAFV3 to efficiently growth in the Cf2Th.CD4.CXCR4; Cf2Th.CXCR4 cell lines at 10:90% cell concentrations, respectively. However after several attempts to infect cultures without CD4 (Cf2Th.CXCR4) have proven unsuccessful, we conclude it is not likely that 05ZAFV3 can adapt to CD4-independence.

c. In vitro phenotypic sensitivity assay

We have established three assays that allow us to monitor the sensitivity and/or resistance of a viral isolate (primary or pseudovirion) to any existing antiretroviral or test compound. These assays include a PBMC-based inhibition assay, a cell line based pseudovirion inhibition assay, and an MTT inhibition assay.


2. Expression and purification of rEnv monomers and trimers (Annwyn Stafne)

Methodologies for the large scale expression and purification of recombinant envelope glycoproteins have been successfully established in our laboratories. Stocks of purified ancestral C gp140 GCN4, 96ZM651gp120-CD5 and gp160-CD5 are available for use in various biochemistry and biological assays to help elucidate the structure-function relationship of this protein.

3. Nonprogressive HIV-1 subtype C infection- viral determinants (Dimitri Tzitzivacos)

We identified six slow progressing HIV-1 positive children, isolated primary virus, and sequenced the full viral genome to identify virus attenuations that may contribute to the altered rates of disease progression. Predicted amino acid sequences for all the HIV-1 proteins were extensively analyzed. Overall, each of the six isolates contained at least one protein with unique insertions, and/or altered functional domains. Results suggest that the slower HIV disease progression in these six children may be attributed to altered protein functions. The biological relevance of these findings needs to be investigated further.

4. The role of HIV-1 subtype C Vpu mediated CD4 down-modulation in disease pathogenesis (Catherine Bell)

HIV-1 subtype C Vpu localizes predominantly to the cell surface, instead of the Golgi apparatus, as has been found for subtype B. The vpu genes for 05ZAFV5 and 05ZAFV15 were codon-optimized, and together with the subtype B vphu were cloned in frame with the EGFP gene into pcDNA3.1. The three VpuEGFP vectors as well as dsRed plasmids targeting various subcellular compartments were each transfected into 293T cells, and observed by confocal microscopy. Cotransfection studies using each Vpu in combination with each of the subcellular compartments are underway.

5. HIV-1 Subtype C Vif Mediated Degradation of APOBEC3G and APOBEC3F (L. Mncube)

This study evaluated the interaction between HIV-1 subtype C Vif and the antiviral factors APOBEC3G and APOBEC3F. The vif genes from isolates 05ZAFV6 and 05ZAFV13 and a control subtype B vif (hvif) were cloned into pDNA3.1/myc- His(-)A. The three vif recombinant plasmids, pcDNA3.1-APOBEC3G-HA and pcDNA3.1- APOBEC3F-V5- 6xHis were each transfected into 293T cells to confirm expression. Cell lysates from co-transfections of each vif-myc with APOBEC3G-HA or APOBEC3F-V5 were then used in anti-myc immunoprecipitation experiments to identify Vif interactions with APOBEC3G and/or APOBEC3F. Results show for the first time that HIV-1 subtype C Vif forms multimers, and is capable of interacting with APOBEC3G and APOBEC3F.

6. Antiviral APOBEC3G genetic variants and impact on HIV infection in an HIV-1 subtype C infected AIDS cohort from South Africa (Audrey Ann Liew, exchange student from Singapore)

APOBEC3G is an important antiviral host factor, and genetic variants could modify APOBEC3G transcription and the functional protein, which may influence HIV-1 transmission and ultimately disease progression to AIDS. However conflicting data has emerged on whether APOBEC3G variants are associated with disease progression. It is thus important to identify APOBEC3G polymorphisms within our population, and evaluate whether these impact on HIV transmission and disease progression. We are currently establishing conventional PCR and sequencing methodologies to identify and compare SNPs and other genetic variants within APOBEC3G from healthy control subjects and AIDS patients.