A single-molecule view on retroviral replication,,

The human immunodeficiency virus (HIV) is a lentivirus that belongs to the family of Retroviridae, and causes the acquired immunodeficiency syndrome (AIDS). In 2009, the number of HIV-infected individuals, estimated at more than 33.3 million (www.unaids.org) which increases annually with two to three million annually. The current treatment of HIV infection is composed of a combination therapy of anti-retroviral agents which efficiently suppresses viral replication. However, due to the seemingly inevitable development of resistance to the existing antiviral cocktails, and the absence of an effective vaccine, the search for new drugs against new targets continues. As HIV has only a limited genomic it hijacks cellular proteins, referred to as co-factors, for the completion of the replication cycle. The laboratory of molecular virology and gene therapy is looking for new cellular cofactors of HIV integrase, the enzyme that integrates the viral cDNA stably into the genome of the infected cell. After reverse transcription of the viral RNA into cDNA in the cytoplasm of the host cell, IN associates the viral DNA with a number of other viral and cellular proteins to form the pre-integration complex (PIC), which is then transported through the nuclear membrane into the nucleus of the host cell. This transport is mediated by the cellular karyopherine transportin-SR2 (TRN-SR2). In the nucleus the PIC is tethered to the chromatin by the binding of the cellular protein LEDGF/p75 (lens epithelium-derived growth factor). Next, the viral DNA is stably integrated into the genome of the host by the catalytic activity of IN, after which new virus particles can be formed. Interactions between these viral proteins and cellular cofactors are attractive targets for the development of novel anti-retroviral agents. Both LEDGF/p75 and TRN-SR2 are at the moment studied by different research laboratories over the world. In 2010 a new class of antiviral molecules was published, the LEDGINs that disrupt the protein-protein interaction between LEDGF/p75 and HIV-1 IN and shows a strong antiviral activity in cell culture. Currently the LEDGINs are further developed for clinical use in collaboration with Pfizer. Despite is has been proven that TRN-SR2 plays a distinct role in the nuclear translocation step, a direct interaction of this protein with IN, has not yet been demonstrated in the context of the PIC in the infected cell. Since this is almost impossible to examine with standard molecular techniques, there is a need for a robust way to study protein-protein interactions of HIV on single-virus and / or single-PIC level in living cells during the early steps of the replication cycle of the virus. The expertise of the laboratory of photochemistry and spectroscopy is the development and application of quantitative fluorescence techniques, particularly techniques for single-molecule spectroscopy and microscopy. In this project, we want to develop and optimize a method to study the early steps of viral replication in a quantitative manner, at single-molecule level, in infected cells using fluorescence imaging. The intended techniques should enable us to characterize the stoichiometry and affinity of the protein-protein interactions between viral and cellular factors in time and space, which will provide crucial information about the interaction of viral proteins with co-factors such as TRN-SR2 and LEDGF / p75 in the search for new inhibitors of HIV replication.status: publishe