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Start of funding 01.07.2005

The Influence of Protein Interaction between HIV-1 Integrase and Reverse Transcriptase for the Initiation of Reverse Transcription

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Searching for compounds against HIV, currently causing one of the most relevant infectious diseases in the world, the viral enzyme reverse transcriptase (RT) is one of the major targets. The RT is active in the early phase of the retroviral replication cycle, and is together with the integrase the major viral component of the preintegration complex, which is responsible for the transport of viral DNA to the nucleus and the integration in the host genome. First experiments with a C-terminal mutant of the integrase showed the lack of protein binding between RT and the mutated integrase despite of unchanged in vitro enzymatic activity of both proteins. However, viruses harbouring the integrase mutant could not perform reverse trancription after infection in cell culture. Aim of the project is to examine wether expression of the C-terminal part of the mutated integrase is able to inhibit viral replication, and therefore to show if protein binding between RT and integrase is essentiell for the initiation of reverse transcription.

Final report:
In the replication cycle of HIV, reverse transcription of the viral RNA into DNA, which can subsequently integrate into the host cell genome, is one of the first major steps. Aim of the project is to investigate the interaction between the HIV-1 proteins integrase (IN) and reverse transcriptase (RT), specifically to determine the importance of the different domains of IN in this interaction. RT can perform reverse transcription in vitro in the absence of IN, but in cell culture the presence of IN is required. A binding site between RT and IN’s c-terminal domain has been identified to be essential for the IN-dependent RT-function. However, also certain mutations outside this region can result in impairment of the RT function. The group of Dr. Chow had created the IN-mutant C130S, which has a Cystein to Serin exchange at position 130 in the core domain of IN. Even though the binding site of IN with RT is not directly affected, the mutation resulted in a loss of reverse transcription in cell culture.

The question which we tried to investigate was, what about the IN is exactly necessary for reverse transcription. It is interesting to know, how the IN facilitates reverse transcription in vivo, whether the physical interaction is necessary or other parts of the IN are required. To investigate the importance of the single domains of IN, these were cloned into a Vpr-fusion constructs. The fusion to Vpr ensures the packaging of the IN-fragments into the virions while conserving a protease cleavage site at the fusion point enabled the IN fragment to be separated from the Vpr during viral maturation. Vectors were produced for the following IN-fragments: C-terminal, ΔC and ΔN and WT-IN. These vectors were co-transfected either with C130S virus or an IN deletion mutant virus – a viral plasmid which had stop mutations at the beginning of the IN sequence to prevent IN expression. Western blot analysis of the collected supernatants could verify, that the IN deletion was successful and all combinations produced virus particles. By extracting the total DNA of cells infected with these viruses and performing quantitative RT-PCR on the early reverse transcription products one should be able to narrow down on the important regions of the IN by determining, which fragments were able to complement the impaired or missing IN best. The infections with IN deleted viruses and fragments of IN should result in a single round of infection only, possibly without any reverse transcription at all, if the fragments don’t provide the required IN function. These experiments allow to determine whether single domains are sufficient to restore revere transcriptase activity and thereby illustrates which IN parts are essential. On the other hand when co-transfecting with the C130S mutant and IN fragments, one can draw conclusions on the mechanism of how the mutation influences the interaction of RT and IN. If providing a healthy core domain results in a complementation of the mutation and the RT activity is restored, then this domain must make a major contribution to the reverse transcription. However if the single c-terminal domain can restore the revere transcription this would indicate that the core-mutation works through some kind of influence on the essential c-terminal part. Therefore this should be investigated in subsequent experiments.