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CRYSTAL STRUCTURES OF THE N-TERMINAL FRAGMENT FROM MOLONEY MURINE LEUKEMIA VIRUS REVERSE TRANSCRIPTASE COMPLEXED WITH NUCLEIC ACID: FUNCTIONAL IMPLICATIONS FOR TEMPLATE-PRIMER BINDING TO THE FINGERS DOMAINCRYSTAL STRUCTURES OF THE N-TERMINAL FRAGMENT FROM MOLONEY MURINE LEUKEMIA VIRUS REVERSE TRANSCRIPTASE COMPLEXED WITH NUCLEIC ACID: FUNCTIONAL IMPLICATIONS FOR TEMPLATE-PRIMER BINDING TO THE FINGERS DOMAIN
Structural highlights
FunctionPOL_MLVMS Gag-Pol polyprotein plays a role in budding and is processed by the viral protease during virion maturation outside the cell. During budding, it recruits, in a PPXY-dependent or independent manner, Nedd4-like ubiquitin ligases that conjugate ubiquitin molecules to Gag, or to Gag binding host factors. Interaction with HECT ubiquitin ligases probably link the viral protein to the host ESCRT pathway and facilitate release. Matrix protein p15 targets Gag and gag-pol polyproteins to the plasma membrane via a multipartite membrane binding signal, that includes its myristoylated N-terminus. Also mediates nuclear localization of the preintegration complex (By similarity). Capsid protein p30 forms the spherical core of the virion that encapsulates the genomic RNA-nucleocapsid complex (By similarity). Nucleocapsid protein p10 is involved in the packaging and encapsidation of two copies of the genome. Binds with high affinity to conserved UCUG elements within the packaging signal, located near the 5'-end of the genome. This binding is dependent on genome dimerization. The aspartyl protease mediates proteolytic cleavages of Gag and Gag-Pol polyproteins during or shortly after the release of the virion from the plasma membrane. Cleavages take place as an ordered, step-wise cascade to yield mature proteins. This process is called maturation. Displays maximal activity during the budding process just prior to particle release from the cell (By similarity). Reverse transcriptase/ribonuclease H (RT) is a multifunctional enzyme that converts the viral dimeric RNA genome into dsDNA in the cytoplasm, shortly after virus entry into the cell. This enzyme displays 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 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 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 primers. 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 the viral genome, matrix protein and integrase. This complex is called the pre-integration complex (PIC). The integrase protein removes 2 nucleotides from each 3' end of the viral DNA, leaving recessed CA OH's at the 3' ends. In the second step that requires cell division, the PIC enters cell nucleus. 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. The last step is viral DNA integration into host chromosome (By similarity). 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 PubMedReverse transcriptase (RT) serves as the replicative polymerase for retroviruses by using RNA and DNA-directed DNA polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process. Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain.,Najmudin S, Cote ML, Sun D, Yohannan S, Montano SP, Gu J, Georgiadis MM J Mol Biol. 2000 Feb 18;296(2):613-32. PMID:10669612[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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