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Solution structure of Simian Foamy Virus (mac) protease
Structural highlights
FunctionPOL_SFV1 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. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedIn contrast to orthoretroviruses, foamy viruses (FVs) express their Pol polyprotein from a separate pol-specific transcript. Only the integrase domain is cleaved off, leading to a protease-reverse transcriptase (PR-RT) protein. We purified the separate PR domain (PRshort) of simian FV from macaques by expressing the recombinant gene in Escherichia coli. Sedimentation analyses and size exclusion chromatography indicate that PRshort is a stable monomer in solution. This allowed us to determine the structure of the PRshort monomer using 1426 experimental restraints derived from NMR spectroscopy. The superposition of 20 conformers resulted in a backbone atom rmsd of 0.55 A for residues Gln8-Leu93. Although the overall folds are similar, the macaque simian FV PRshort reveals significant differences in the dimerization interface relative to other retroviral PRs, such as HIV-1 (human immunodeficiency virus type 1) PR, which appear to be rather stable dimers. Especially the flap region and the N- and C-termini of PRshort are highly flexible. Neglecting these regions, the backbone atom rmsd drops to 0.32 A, highlighting the good definition of the central part of the protein. To exclude that the monomeric state of PRshort is due to cleaving off the RT, we purified the complete PR-RT and performed size exclusion chromatography. Our data show that PR-RT is also monomeric. We thus conclude adoption of a monomeric state of PR-RT to be a regulatory mechanism to inhibit PR activity before virus assembly in order to reduce packaging problems. Dimerization might therefore be triggered by additional viral or cellular factors. The solution structure of the simian foamy virus protease reveals a monomeric protein.,Hartl MJ, Wohrl BM, Rosch P, Schweimer K J Mol Biol. 2008 Aug 1;381(1):141-9. Epub 2008 Jun 3. PMID:18597783[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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