4e7h | pdb_00004e7h
PFV intasome prior to 3'-processing, Apo form (UI-Apo)
Structural highlights
Function[POL_FOAMV] The aspartyl protease activity mediates proteolytic cleavages of Gag and Pol polyproteins. The reverse transcriptase (RT) activity converts the viral RNA genome into dsDNA in the cytoplasm, shortly after virus entry into the cell (early reverse transcription) or after proviral DNA transcription (late reverse transcription). RT consists of a DNA polymerase activity that can copy either DNA or RNA templates, and a ribonuclease H (RNase H) activity that cleaves the RNA strand of RNA-DNA heteroduplexes in a partially processive 3' to 5' endonucleasic mode. Conversion of viral genomic RNA into dsDNA requires many steps. A tRNA-Lys1,2 binds to the primer-binding site (PBS) situated at the 5'-end of the viral RNA. RT uses the 3' end of the tRNA primer to perform a short round of RNA-dependent minus-strand DNA synthesis. The reading proceeds through the U5 region and ends after the repeated (R) region which is present at both ends of viral RNA. The portion of the RNA-DNA heteroduplex is digested by the RNase H, resulting in a ssDNA product attached to the tRNA primer. This ssDNA/tRNA hybridizes with the identical R region situated at the 3' end of viral RNA. This template exchange, known as minus-strand DNA strong stop transfer, can be either intra- or intermolecular. RT uses the 3' end of this newly synthesized short ssDNA to perform the RNA-dependent minus-strand DNA synthesis of the whole template. RNase H digests the RNA template except for a polypurine tract (PPT) situated at the 5'-end and near the center of the genome. It is not clear if both polymerase and RNase H activities are simultaneous. RNase H probably can proceed both in a polymerase-dependent (RNA cut into small fragments by the same RT performing DNA synthesis) and a polymerase-independent mode (cleavage of remaining RNA fragments by free RTs). Secondly, RT performs DNA-directed plus-strand DNA synthesis using the PPT that has not been removed by RNase H as primer. PPT and tRNA primers are then removed by RNase H. The 3' and 5' ssDNA PBS regions hybridize to form a circular dsDNA intermediate. Strand displacement synthesis by RT to the PBS and PPT ends produces a blunt ended, linear dsDNA copy of the viral genome that includes long terminal repeats (LTRs) at both ends (By similarity). Integrase catalyzes viral DNA integration into the host chromosome, by performing a series of DNA cutting and joining reactions. This enzyme activity takes place after virion entry into a cell and reverse transcription of the RNA genome in dsDNA. The first step in the integration process is 3' processing. This step requires a complex comprising at least the viral genome, matrix protein, and integrase. This complex is called the pre-integration complex (PIC). The integrase protein removes 2 nucleotides from the 3' end of the viral DNA right (U5) end, leaving the left (U3) intact. In the second step, the PIC enters cell nucleus. This process is mediated through the integrase and allows the virus to infect both dividing (nuclear membrane disassembled) and G1/S-arrested cells (active translocation), but with no viral gene expression in the latter. In the third step, termed strand transfer, the integrase protein joins the previously processed 3' ends to the 5' ends of strands of target cellular DNA at the site of integration. It is however not clear how integration then proceeds to resolve the asymmetrical cleavage of viral DNA (By similarity). Publication Abstract from PubMedRetroviral integrase (IN) is responsible for two consecutive reactions, which lead to insertion of a viral DNA copy into a host cell chromosome. Initially, the enzyme removes di- or trinucleotides from viral DNA ends to expose 3'-hydroxyls attached to the invariant CA dinucleotides (3'-processing reaction). Second, it inserts the processed 3'-viral DNA ends into host chromosomal DNA (strand transfer). Herein, we report a crystal structure of prototype foamy virus IN bound to viral DNA prior to 3'-processing. Furthermore, taking advantage of its dependence on divalent metal ion cofactors, we were able to freeze trap the viral enzyme in its ground states containing all the components necessary for 3'-processing or strand transfer. Our results shed light on the mechanics of retroviral DNA integration and explain why HIV IN strand transfer inhibitors are ineffective against the 3'-processing step of integration. The ground state structures moreover highlight a striking substrate mimicry utilized by the inhibitors in their binding to the IN active site and suggest ways to improve upon this clinically relevant class of small molecules. 3'-Processing and strand transfer catalysed by retroviral integrase in crystallo.,Hare S, Maertens GN, Cherepanov P EMBO J. 2012 May 11. doi: 10.1038/emboj.2012.118. PMID:22580823[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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