Intramolecular integration within Moloney murine leukemia virus DNA

By screening a library of unintegrated, circular Moloney murine leukemia virus (M-MuLV) DNA cloned in lambda phage, we found that approximately 20% of the M-MuLV DNA inserts contained internal sequence deletions or inversions. Restriction enzyme mapping demonstrated tht the deleted segments frequently abutted a long terminal repeat (LTR) sequence, whereas the inverted segments were usually flanked by LTR sequences, suggesting that many of the variants arose as a consequence of M-MuLV DNA molecules integrating within their own DNA. Nucleotide sequencing also suggested that most of the variant inserts were generated by autointegration. One of the recombinant M-MuLV DNA inserts contained a large inverted repeat of a unique M-MuLV sequence abutting an LTR. This molecule was shown by nucleotide sequencing to have arisen by an M-MuLV DNA Molecule integrating within a second M-MuLV DNA molecule before cloning. The autointegrated M-MuLV DNA had generally lost two base pairs from the LTR sequence at each junction with target site DNA, whereas a four-base-pair direct repeat of target site DNA flanked the integrated viral DNA. Nucleotide sequencing of preintegration target site DNA showed that this four-base-pair direct repeat was present only once before integration and was thus reiterated by the integration event. The results obtained from the autointegrated clones were supported by nucleotide sequencing of the host-virus junction of two cloned M-MuLV integrated proviruses obtained from infected rat cells. Detailed analysis of the different unique target site sequences revealed no obvious common features

Destruction of Contingent Interests by Termination of a Trust

Destruction of Contingent Interests by Termination of a Trus

Dynein Regulators Are Important for Ecotropic Murine Leukemia Virus Infection

Indexación: Web of Science.During the early steps of infection, retroviruses must direct the movement of the viral genome into the nucleus to complete their replication cycle. This process is mediated by cellular proteins that interact first with the reverse transcription complex and later with the preintegration complex (PIC), allowing it to reach and enter the nucleus. For simple retroviruses, such as murine leukemia virus (MLV), the identities of the cellular proteins involved in trafficking of the PIC in infection are unknown. To identify cellular proteins that interact with the MLV PIC, we developed a replication-competent MLV in which the integrase protein was tagged with a FLAG epitope. Using a combination of immunoprecipitation and mass spectrometry, we established that the microtubule motor dynein regulator DCTN2/p50/dynamitin interacts with the MLV preintegration complex early in infection, suggesting a direct interaction between the incoming viral particles and the dynein complex regulators. Further experiments showed that RNA interference (RNAi)-mediated silencing of either DCTN2/p50/dynamitin or another dynein regulator, NudEL, profoundly reduced the efficiency of infection by ecotropic, but not amphotropic, MLV reporters. We propose that the cytoplasmic dynein regulators are a critical component of the host machinery needed for infection by the retroviruses entering the cell via the ecotropic envelope pathway. IMPORTANCE Retroviruses must access the chromatin of host cells to integrate the viral DNA, but before this crucial event, they must reach the nucleus. The movement through the cytoplasm-a crowded environment where diffusion is slow-is thought to utilize retrograde transport along the microtubule network by the dynein complex. Different viruses use different components of this multi-subunit complex. We found that the preintegration complex of murine leukemia virus (MLV) interacts with the dynein complex and that regulators of this complex are essential for infection. Our study provides the first insight into the requirements for retrograde transport of the MLV preintegration complex.http://jvi.asm.org/content/90/15/689

Nuclear matrix protein Matrin 3 is a regulator of ZAP-mediated retroviral restriction

Background Matrin 3 is a nuclear matrix protein involved in multiple nuclear processes. In HIV-1 infection, Matrin 3 serves as a Rev cofactor important for the cytoplasmic accumulation of HIV-1 transcripts. ZAP is a potent host restriction factor of multiple viruses including retroviruses HIV-1 and MoMuLV. In this study we sought to further characterize Matrin 3 functions in the regulation of HIV gene expression. Results Here we describe a function for Matrin 3 as a negative regulator of the ZAP-mediated restriction of retroviruses. Mass spectrometry analysis of Matrin 3-associated proteins uncovered interactions with proteins of the ZAP degradation complex, DDX17 and EXOSC3. Coimmunoprecipitation studies confirmed Matrin 3 associations with DDX17, EXOSC3 and ZAP, in a largely RNA-dependent manner, indicating that RNA is mediating the Matrin 3 interactions with these components of the ZAP degradation complex. Silencing Matrin 3 expression caused a remarkably enhanced ZAP-driven inhibition of HIV-1 and MoMuLV luciferase reporter viruses. This effect was shared with additional nuclear matrix proteins. ZAP targets multiply-spliced HIV-1 transcripts, but in the context of Matrin 3 suppression, this ZAP restriction was broadened to unspliced and multiply-spliced RNAs. Conclusions Here we reveal an unprecedented role for a nuclear matrix protein, Matrin 3, in the regulation of ZAP’s antiretroviral activity. Suppressing Matrin 3 powers a heightened and broader ZAP restriction of HIV-1 gene expression. This study suggests that this ZAP regulatory mechanism is shared with additional nuclear matrix proteins

Comment on “Late Cenozoic sea level and the rise of modern rimmed atolls” by Toomey et al. (2016), Palaeogeography, Palaeoclimatology, Palaeoecology 451: 73–83

© 2016 Elsevier B.V. Toomey et al. (2016) present a credible new model to explain variations in the formation of Pacific Ocean atolls through the Late Cenozoic, thus accounting for contemporary differences in atoll morphology. While we do not contradict the primary influences of dissolution and sedimentation processes, we suggest that the influence of submarine landsliding should not be ignored. Multiple slope failures exist on the flanks of many atolls. When large submarine landslides affected a significant portion of a palaeo-atoll rim, this had the potential to open up an atoll lagoon allowing much freer circulation between the lagoon and the ocean. Our atoll flank collapse model therefore provides an additional mechanism for an enclosed atoll lagoon to become a ‘leaking bucket’ system and an atoll more likely to evolve into the ‘empty bucket’ form with later sea-level rise. Submarine landsliding adds additional support for the new model of atoll formation proposed by Toomey et al

Host proteins interacting with the Moloney murine leukemia virus integrase: Multiple transcriptional regulators and chromatin binding factors

Background 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. Results 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. Conclusion 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

Isolation of suppressor genes that restore retrovirus susceptibility to a virus-resistant cell line

BACKGROUND: Genetic selections in mammalian cell lines have recently been developed for the isolation of mutant cells that are refractory to infection by retroviruses. These selections have been used to recover lines that block early postentry stages of infection, either before reverse transcription or before nuclear entry. The mechanisms of action of these blocks remain unknown. RESULTS: We have devised a method for the selection of genes from cDNA libraries that suppress the block to virus infection, and so restore virus susceptibility. The protocol involves the transformation of pools of resistant cells by cDNA expression libraries, followed by the selection for rare virus-sensitive cells, using multiple rounds of selection after infection by marked viral vector genomes. The suppressor genes were then recovered from these virus sensitive cells, and their ability to restore virus susceptibility was confirmed by reintroduction of these cDNAs into the resistant line. CONCLUSIONS: The identities of these genes provide insights into the mechanism of virus resistance and will help to define new pathways used during retrovirus infection. The methods for gene isolation developed here will also permit the identification of similar suppressors that modify or override other recently identified virus resistance genes

Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities

The reverse transcriptase of Moloney murine leukemia virus, like that of all retroviruses, exhibits a DNA polymerase activity capable of synthesis on RNA or DNA templates and an RNase H activity with specificity for RNA in the form of an RNA.DNA hybrid. We have generated a library of linker insertion mutants of the Moloney murine leukemia virus enzyme expressed in bacteria and assayed these mutants for both enzymatic activities. Those mutations affecting the DNA polymerase activity were clustered in the 5'-proximal two-thirds of the gene, and those affecting RNase H were in the remaining 3' one-third. Based on these maps, plasmids were made that expressed each one of the domains separately; assays of the proteins encoded by these plasmids showed that each domain exhibited only the expected activity

Tsunamigenic slope failures: the Pacific Islands ‘blind spot’?

© 2015, Springer-Verlag Berlin Heidelberg. We discuss issues related to a recognised shortcoming in existing tsunami hazard assessments for Pacific Island Countries and Territories (PICTs), that of tsunamigenic slope failures (TSFs). Currently, TSFs are most likely underrepresented as sources in existing tsunami databases for two key reasons. First, relatively low magnitude earthquakes associated with subduction zones are generally assigned as the tsunamigenic source, as opposed to the TSFs they generate. A reassessment of such ‘anomalous tsunamis’ may yield clues that serve to reassign their tsunamigenic source. Second, there are thousands of oceanic islands and seamounts scattered across the Pacific and flank collapse of volcanic edifices such as these is a largely unquantified tsunamigenic threat. However, while it is now possible to model such TSFs, this is unlikely to happen in the near future because of the lack of detailed bathymetry and landslide mass data. Recent developments in the identification of past tsunamis in the Pacific Islands have developed a unique range of indicators that can be used for identifying such events. These are geological, oral tradition and archaeological components that include, but are not limited to, a modified Darwinian model of atoll formation, coastal megaclasts, oral traditions of vanished islands and giant waves, and the abandonment of prehistoric coastal sites. As such, the most logical way forward is to use the multiple indicators available to us to identify evidence of past tsunamis