2lsn
From Proteopedia
Solution structure of PFV RNase H domain
Structural highlights
FunctionPOL_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 PubMedABSTRACT: BACKGROUND: The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity. RESULTS: The solution structure of PFV RNase H shows that it contains a mixed five-stranded beta-sheet, which is sandwiched by four alpha-helices (A-D), including the C-helix, on one side and one alpha-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding. CONCLUSIONS: The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding. The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding.,Leo B, Schweimer K, Rosch P, Hartl MJ, Wohrl BM Retrovirology. 2012 Sep 10;9:73. PMID:22962864[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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