Research Summaries

Waldemar Popik
Associate Professor

One of the forms of the innate immunity against HIV is mediated by cytidine deaminases APOBEC3G and APOBEC3F. Although the enzymatic editing of the HIV reverse transcripts is believed to be the primary antiretroviral function of these cellular deaminases, additional nonenzymatic antiviral activities have been suggested for APOBEC3G.

APOBEC3G is expressed in T lymphocytes and macrophages, the main targets of HIV. However, during virus infection, the levels of cellular APOBEC3G can be drastically reduced due to the presence of viral protein Vif. In addition, sequestration of APOBEC3G in an inactive high molecular mass ribonucleoprotein complex may render the cells susceptible to HIV infection. Thus, the inhibition of the interaction between APOBEC3G and Vif or, alternatively, increasing the cellular levels of APOBEC3G or mobilization of the inactive high mass APOBEC3G complexes and release of the active low mass form of APOBEC3G may restore the antiretroviral status of the cell.

The observation that the APOBEC3G 186R polymorphism, which is frequently found in African Americans (37%) but rare in European Americans (5%), may be associated with accelerated progression to AIDS, suggests an urgent need for the development of alternative therapeutic strategies that may be effective in protecting people expressing inactive APOBEC3G.

We and others have demonstrated that the antiretroviral activity of APOBEC3G requires its packaging into assembling HIV virions through the interaction with the Gag nucleocapsid protein NC and viral RNA. However, this process is strongly limited in the presence of the viral accessory protein Vif, which binds to and targets APOBEC3G for degradation by proteasomes. Thus, finding means of escaping the Vif-mediated degradation has become a challenge to developing an effective APOBEC3G-based antiretroviral therapy.

We have recently demonstrated that H9 T cells secrete vesicles with encapsidated APOBEC3G. Biochemical characterization of the released vesicles (sucrose gradient floatation and Western blot analysis) and immunogold electron microscopy confirmed that these vesicles have features characteristic for exosomes. Interestingly, we have found that H9 exosomes show an enzymatic activity when incubated with a synthetic oligonucleotide, resulting in deamination of a third cytidine in the CCC motif which is specifically recognized and targeted by APOBEC3G.

Using H9 exosomes (APO-exosomes) labeled with green fluorescent lipid PKH67, we have shown that APO-exosomes were internalized by a variety of cells including HeLa, Jurkat T and promonocytic THP-1 cells. Further analysis showed that APO-exosomes severely restricted replication of Vif-deficient NL4-3 HIV-1 when incubated with infected Jurkat and THP-1 cells. These results suggest that APOBEC3G in exosomes was delivered to target cells and more importantly was able to perform its antiretroviral activity.

Our current research is focused on the elucidation of the molecular mechanism(s) for targeting APOBEC3G into exosomes and evaluating the potential of exosome-encapsidated APOBEC3G as a novel approach in anti-HIV therapy.