Mechanism of HIV-1 gene expression. We study the mechanism of LTR-mediated viral transcription. Appropriate in vitro and in vivo assay systems have been developed for these studies, including the evolutionary analysis of mutated viruses. We analyzed the LTR promoter activity for the different HIV-1 subtypes. The mechanism of transcriptional activation by the viral Tat protein through the TAR hairpin motif has been addressed at the molecular level by different approaches. More recently, we have initiated studies on the mechanism of proviral latency. Other aspects of viral gene expression, such as the regulation of mRNA splicing and polyadenylation are also being addressed. HIV-1 RNA structure and function. Studies have focused on the untranslated leader that contains many replication signals, e.g. for RNA dimerization and RNA packaging. A riboswitch model was proposed to differentiate between the gene expression (translation) and virus-specific activities (virion packaging) of the HIV-1 RNA genome. We reported that the 9kb HIV-1 RNA circularizes by means of a 5'-3' basepairing interaction. We also have a special interest in the nucleotide-composition and codon usage of
retroviruses. We also study the initiation process of HIV-1 reverse transcription, which requires the interaction between the primer-binding site (PBS) and the cellular tRNAlys3 primer and the Reverse Transcriptase enzyme. Virus evolution and drug-resistance. We study virus evolution in diverse settings, e.g. the selection of drug-resistant virus variants and the selection of revertant viruses from replication-impaired HIV-1 mutants. Drug resistance was studied for the RT inhibitor 3TC (lamivudine) and we describede for the first time a fitness loss of the mutated RT enzyme and virus. Other studies focus on the mechanism that provides resistance to the entry inhibitor T20 (Fuzeon), we e.g. reported the first drug-dependent HIV-1 variant and RNAi-resistance (see below). RNA interference as antiviral therapy. HIV-1 replication in cell culture can be effectively blocked by intracellular expresion of a single shRNA inhibitor that induces RNA interference (RNAi) against HIV-1 or a critical cellular co-factor. However, the virus can escape as was demonstrated in massive evolution studies that described different escape routes. We have succesfully blocked viral escape by a
combinatorial RNAi approach that is now being tested in a humanized mouse model as pre-clinical test system. A lentivirus-mediated gene therapy is being developed based on this system. Basic aspects of the virus-RNAi interaction, e.g. the presence of viral RNAi suppressors, are also being studied. We recently described a novel Dicer-independent RNAi pathway. Novel vaccine strategies. Live-attenuated virus vaccines have shown to be much more effective than any other AIDS vaccine approach. However, the attenuated virus may revert over time to a virulent and pathogenic phenotype, and live attenuated HIV vaccines are therefore considered to be unsafe. To improve the safety, we have constructed a conditional-live virus in which the doxycycline (dox)-controlled gene expression system (Tet-On system) was incorporated. Upon vaccination, replication of this virus can be limited to the time required to induce a protective immune response by transient administration of dox, which will prevent the evolution of a more pathogenic virus. Promising results have recently been obtained in the SIV-macaque model. Spin-off is formed by the evolution of imporved Tet-On systems (collaboration with dr.
A.T. Das). Patient related virus studies. Based on the availability of relevant patient samples, e.g. as member of the Amsterdam Cohort Studies on HIV-AIDS, we are performing several clinically relevant virology studies. This includes the description of HIV-1 superinfections, novel HIV-1 subtype variants, HBV double infections, and the development of ultra-sensitive intracellular HIV-1 RNA detection assays.