EBP1, a novel host factor involved in primer binding site-dependent restriction of moloney murine leukemia virus in embryonic cells.
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ABSTRACT:
SUBMITTER: Wang GZ
PROVIDER: S-EPMC3911593 | biostudies-literature | 2014 Feb
REPOSITORIES: biostudies-literature
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Project description:Retroviral reverse transcriptase (RT) of Moloney murine leukemia virus (MoMLV) is expressed in the form of a large Gag-Pol precursor protein by suppression of translational termination in which the maximal efficiency of stop codon read-through depends on the interaction between MoMLV RT and peptidyl release factor 1 (eRF1). Here, we report the crystal structure of MoMLV RT in complex with eRF1. The MoMLV RT interacts with the C-terminal domain of eRF1 via its RNase H domain to sterically occlude the binding of peptidyl release factor 3 (eRF3) to eRF1. Promotion of read-through by MoMLV RNase H prevents nonsense-mediated mRNA decay (NMD) of mRNAs. Comparison of our structure with that of HIV RT explains why HIV RT cannot interact with eRF1. Our results provide a mechanistic view of how MoMLV manipulates the host translation termination machinery for the synthesis of its own proteins.
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Project description:Spl574 MLV (murine leukemia virus) is a variant of Moloney ecotropic MLV (MoMLV) that is cytopathic in Mus dunni cells and restricted by other mouse cells. Its host range and cytopathicity are due to a mutation, S82F, at a site critical for binding to the CAT-1 receptor. To identify residues that affect affinity for receptor variants, virus with S82F was passed in restrictive cells. The env genes of the adapted viruses contained 18 novel mutations, including one, E114G, present in 6 of 30 sequenced envs. MoMLV-E114G efficiently infected all mouse cells as well as ecotropic MLV resistant Chinese hamster cells. Virus with E114G and S82F induced large multinucleated syncytia in NIH 3T3 and SC-1 cells as well as M. dunni cells. Inoculation of Mo-S82F,E114G into mice produced lymphomas typical of MoMLV. Residues at env position 114 are thus important determinants of host range, and E114G suppresses host range restriction due to S82F, but does not affect S82F-governed cytopathicity.
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Project description:In the first step of retroviral integration, integrase cleaves the linear viral DNA within its long terminal repeat (LTR) immediately 3' to the CA dinucleotide step, resulting in a reactive 3' OH on one strand and a 5' two base overhang on the complementary strand. In order to investigate the structural properties of the 3' end processing site within the Moloney murine leukemia virus (MMLV) LTR d(TCTTTCATT), a host-guest crystallographic method was employed to determine the structures of four self-complementary 16 bp oligonucleotides including LTR sequences (underlined), d(TTTCATTGCAATGAAA), d(CTTTCATTAATGAAAG), d(TCTTTCATATGAAAGA) and d(CACAATGATCATTGTG), the guests, complexed with the N-terminal fragment of MMLV reverse transcriptase, the host. The structures of the LTR-containing oligonucleotides were compared to those of non-LTR oligonucleotides crystallized in the same lattice. Properties unique to the CA dinucleotide step within the LTR sequence, independent of its position from the end of the duplex, include a positive roll angle and negative slide value. This propensity for the CA dinucleotide step within the MMLV LTR sequence to adopt only positive roll angles is likely influenced by the more rigid, invariable 3' and 5' flanking TT dinucleotide steps and may be important for specific recognition and/or cleavage by the MMLV integrase.
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Project description:A critical step for retroviral replication is the stable integration of the provirus into the genome of its host. The viral integrase protein is key in this essential step of the retroviral life cycle. Although the basic mechanism of integration by mammalian retroviruses has been well characterized, the factors determining how viral integration events are targeted to particular regions of the genome or to regions of a particular DNA structure remain poorly defined. Significant questions remain regarding the influence of host proteins on the selection of target sites, on the repair of integration intermediates, and on the efficiency of integration.We describe the results of a yeast two-hybrid screen using Moloney murine leukemia virus integrase as bait to screen murine cDNA libraries for host proteins that interact with the integrase. We identified 27 proteins that interacted with different integrase fusion proteins. The identified proteins include chromatin remodeling, DNA repair and transcription factors (13 proteins); translational regulation factors, helicases, splicing factors and other RNA binding proteins (10 proteins); and transporters or miscellaneous factors (4 proteins). We confirmed the interaction of these proteins with integrase by testing them in the context of other yeast strains with GAL4-DNA binding domain-integrase fusions, and by in vitro binding assays between recombinant proteins. Subsequent analyses revealed that a number of the proteins identified as Mo-MLV integrase interactors also interact with HIV-1 integrase both in yeast and in vitro.We identify several proteins interacting directly with both MoMLV and HIV-1 integrases that may be common to the integration reaction pathways of both viruses. Many of the proteins identified in the screen are logical interaction partners for integrase, and the validity of a number of the interactions are supported by other studies. In addition, we observe that some of the proteins have documented interactions with other viruses, raising the intriguing possibility that there may be common host proteins used by different viruses. We undertook this screen to identify host factors that might affect integration target site selection, and find that our screens have generated a wealth of putative interacting proteins that merit further investigation.
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Project description:The matrix protein (MA) of the Moloney murine leukemia virus (M-MuLV) was found to interact with IQGAP1, a prominent regulator of the cytoskeleton. Mutational studies defined residues of MA critical for the interaction, and tests of viruses carrying MA mutations revealed a near-perfect correlation between binding and virus replication. The replication-defective mutants showed defects in both early and late stages of the life cycle. Four viable second-site revertant viruses were isolated from three different replication-defective parental mutants, and in all cases the interaction with IQGAP1 was restored by the suppressor mutations. The interaction of MA and IQGAP1 was readily detected in vitro and in vivo. Virus replication was potently inhibited by a C-terminal fragment of IQGAP1, and impaired by RNAi knockdown of IQGAP1 and 2. We suggest that the IQGAPs link the virus to the cytoskeleton for trafficking both into and out of the cell.
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Project description:Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary to findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta-chain, the immunoglobulin kappa light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the primer binding site was altered to match that of a new primer species, tRNA1,2Lys. In addition, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant.
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