Imaging the early steps of HIV-1 replication by labelling integrase and viral DNA

HIV-1 requires various interactions with cellular cofactors to complete its viral replication cycle. In the first place, viral entry involves the interaction of the viral envelope protein with the host cell CD4-receptor and the CXCR4 or CCR5 co-receptor, resulting in membrane fusion and the release of the viral cone into the cytoplasm of the host cell. During the transport to the nucleus, the viral RNA is converted into double-stranded viral DNA (vDNA) by the viral reverse transcriptase (RT) together with the initiation of viral uncoating. At the nuclear pore complex, the viral capsid protein interacts with different nucleoporins, components of the nuclear pore complex, in order to gain access to the nucleus. Additionally, different cellular import factors, including importin-7, importin-a1, importin-a3 and transportin-SR2 (TRN-SR2) have been implicated in HIV-1 nuclear import. In 2008, TRN-SR2 was identified as an HIV-1 cofactor in two independent genome-wide RNAi screens and as an interaction partner of HIV-1 integrase (IN) in a yeast-two-hybrid screen. After nuclear entry, the vDNA is tethered to the host cell genome by the cellular protein LEDGF/p75, where it is integrated by the viral IN. These different steps of the early HIV-1 replication cycle are all interconnected and precisely timed. In the end, only a small population of the viruses entering a host cell performs all these steps correctly leading to a persistent infection. Single-virus fluorescence techniques, based on the fluorescent labelling of a viral protein, allows us to monitor specific steps of the replication cycle in a cellular context, and in a highly sensitive and selective way. This provides information about the intracellular location, interaction partners and dynamics, even in living cells. In chapter 3, I focussed on a disease-associated TRN-SR2 mutant and its effect on HIV-1 replication. TRN-SR2, encoded by the TNPO3 gene, imports essential pre-mRNA splicing factors, including ASF/SF2, SC35 and CPSF6. An adenosine deletion in the stop codon of TRN-SR2 was found to be the genetic cause of the muscle disease limb girdle muscular dystrophy 1F (LGMD1F). Due to a frameshift, resulting from the adenosine deletion, patients with LGMD1F contain an extended TRN-SR2 protein, adding fifteen extra amino acids at its carboxyl-terminal end. Nuclear translocation of the elongated TRN-SR2 is affected, although the exact mechanism by which this mutation leads to LGMD1F is currently still unknown. Because of the essential role of TRN-SR2 in HIV-1 nuclear import, the effect of this mutant on HIV-1 replication and nuclear import was studied. This was done using several techniques including fluorescently labelled viruses containing IN coupled to eGFP. We demonstrate that HIV-1 replication is hampered in cell lines containing the mutated TRN-SR2 variant and even more in primary cells from patients with LGMD1F, although this block could not be pinpointed to the stage of nuclear import. The exact mechanism by which this mutation inhibits HIV-1 infection still needs to be elucidated. The use of the integrase labelled viruses enables us to study single viruses, but it does not allow us to identify the small pool of infectious particles within the large background of non-infectious ones. Since successful viral infection is determined by the integration of the vDNA into the host chromosome, I focussed in chapter 4 of my thesis on a novel HIV-1 DNA labelling method. Numerous HIV-1-specific and non-specific DNA labelling strategies have already been developed, however most techniques cannot be combined with naturally occurring fluorescent proteins, such as GFP. Hence, we opted for the development of an HIV-1 DNA labelling strategy that enabled the combination with particles containing the fluorescent-tagged HIV-1 integrase. This new technique is based on the azide-alkyne cycloaddition reaction also referred to as click chemistry. Nucleosides containing a clickable triple bond are provided during infection and are incorporated by RT. Subsequently, the nucleoside analogues are coupled to an organic dye leading to fluorescently labelled vDNA. 5-ethynyl-2'-deoxyuridine (EdU), a commercial available nucleoside analogue, was already used in HIV-1 research, however, this resulted in a substantial background labelling of the host cell itself. Therefore, we attempted to develop new nucleoside analogues, which would selectively and efficiently label the HIV-1 DNA. Seven propargylated nucleoside analogues were synthesised and tested for their lack of cytotoxicity and virus inhibition, RT-specific primer extension and incorporation kinetics in vitro, and the capacity to stain HIV-1 DNA. One of the nucleoside analogues (A5) was specifically incorporated by HIV-1 RT, however, no efficient incorporation in vitro or vDNA staining during infection was detected. Two other analogues (A3 and A6) were incorporated in vitro by HIV-1 RT and human mitochondrial DNA polymerase γ, and did enable specific HIV-1 DNA labelling, although not very efficient. Additionally, A3 supported mitochondria-specific DNA labelling, and hence can be used to visualise mitochondrial DNA synthesis. Altogether, no efficiently incorporated, HIV-1 RT-specific nucleoside analogue selectively labelling the HIV-1 DNA could be identified. However, these results provide information for further chemical refinement necessary to develop more efficient HIV-1 DNA labels.

Jaar van publicatie:2019

Toegankelijkheid:Closed