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Structural studies of the catalytic core of the primate foamy virus (PFV-1) integrase.

ABSTRACT: Retroviral integrases are vital enzymes in the viral life cycle and thus are important targets for antiretroviral drugs. The structure of the catalytic core domain of the integrase from human foamy virus, which is related to HIV-1, has been solved. The structure of the protein is presented in two different crystal forms, each containing several molecules in the asymmetric unit, with and without the essential manganese or magnesium ion, and the structures are compared in detail. This allows regions of high structural variability to be pinpointed, as well as the effect of divalent cations on the conformation of the catalytic site.

SUBMITTER: Rety S

PROVIDER: S-EPMC2917282 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Structural studies of the catalytic core of the primate foamy virus (PFV-1) integrase.

Réty Stéphane S Reaeábková Lenka L Dubanchet Barbara B Silhán Jan J Legrand Pierre P Lewit-Bentley Anita A

Acta crystallographica. Section F, Structural biology and crystallization communications 20100727 Pt 8

Retroviral integrases are vital enzymes in the viral life cycle and thus are important targets for antiretroviral drugs. The structure of the catalytic core domain of the integrase from human foamy virus, which is related to HIV-1, has been solved. The structure of the protein is presented in two different crystal forms, each containing several molecules in the asymmetric unit, with and without the essential manganese or magnesium ion, and the structures are compared in detail. This allows regio ...[more]

PMID: 20693659

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In vitro fidelity of the prototype primate foamy virus (PFV) RT compared to HIV-1 RT.

Project description:We compared the in vitro fidelity of wild-type human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) and the prototype foamy virus (PFV) RT. Both enzymes had similar error rates for single nucleotide substitutions; however, PFV RT did not appear to make errors at specific hotspots, like HIV-1 RT. In addition, PFV RT made more deletions and insertions than HIV-1 RT. Although the majority of the missense errors made by HIV-1 RT and PFV RT are different, relatively few of the mutations caused by either enzyme can be explained by a misalignment/slippage mechanism. We suggest that the higher polymerase activity of PFV RT could contribute to the ability of the enzyme to jump to the same or a different template.

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Functional and structural characterization of the integrase from the prototype foamy virus.

Project description:Establishment of the stable provirus is an essential step in retroviral replication, orchestrated by integrase (IN), a virus-derived enzyme. Until now, available structural information was limited to the INs of human immunodeficiency virus type 1 (HIV-1), avian sarcoma virus (ASV) and their close orthologs from the Lentivirus and Alpharetrovirus genera. Here, we characterized the in vitro activity of the prototype foamy virus (PFV) IN from the Spumavirus genus and determined the three-dimensional structure of its catalytic core domain (CCD). Recombinant PFV IN displayed robust and almost exclusively concerted integration activity in vitro utilizing donor DNA substrates as short as 16 bp, underscoring its significance as a model for detailed structural studies. Comparison of the HIV-1, ASV and PFV CCD structures highlighted both conserved as well as unique structural features such as organization of the active site and the putative host factor binding face. Despite possessing very limited sequence identity to its HIV counterpart, PFV IN was sensitive to HIV IN strand transfer inhibitors, suggesting that this class of inhibitors target the most conserved features of retroviral IN-DNA complexes.

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Improved primate foamy virus vectors and packaging constructs.

Project description:Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5' splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5' SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5' SD or an unrelated intron sequence was substituted for the FV 5' untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.

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Molecular dissection of the prototype foamy virus (PFV) RNA 5'-UTR identifies essential elements of a ribosomal shunt.

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Zoonotic infection of Brazilian primate workers with New World simian foamy virus.

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Lnc-RP5 Regulates the miR-129-5p/Notch1/PFV Internal Promoter Axis to Promote the Expression of the Prototype Foamy Virus Transactivator Tas.

Project description:Prototype foamy virus (PFV) is a unique retrovirus that infects animals and humans and does not cause clinical symptoms. Long noncoding RNAs (lncRNAs) are believed to exert multiple regulatory functions during viral infections. Previously, we utilized RNA sequencing (RNA-seq) to characterize and identify the lncRNA lnc-RP5-1086D14.3.1-1:1 (lnc-RP5), which is markedly decreased in PFV-infected cells. However, little is known about the function of lnc-RP5 during PFV infection. In this study, we identified lnc-RP5 as a regulator of the PFV transcriptional transactivator (Tas). Lnc-RP5 enhanced the activity of the PFV internal promoter (IP). The expression of PFV Tas was found to be promoted by lnc-RP5. Moreover, miR-129-5p was found to be involved in the lnc-RP5-mediated promotion of PFV IP activity, while the Notch1 protein suppressed the activity of PFV IP and the expression of Tas. Our results demonstrate that lnc-RP5 promotes the expression of PFV Tas through the miR-129-5p/Notch1/PFV IP axis. This work provides evidence that host lncRNAs can manipulate PFV replication by employing miRNAs and proteins during an early viral infection.

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Structural and evolutionary analysis of an orangutan foamy virus.

Project description:The full-length proviral genome of a foamy virus infecting a Bornean orangutan was amplified, and its sequence was analyzed. Although the genome showed a clear resemblance to other published foamy virus genomes from apes and monkeys, phylogenetic analysis revealed that simian foamy virus SFVora was evolutionarily equidistant from foamy viruses from other hominoids and from those from Old World monkeys. This finding suggests an independent evolution within its host over a long period of time.

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Solution conformations of prototype foamy virus integrase and its stable synaptic complex with U5 viral DNA.

Project description:Using small-angle X-ray and neutron scattering (SAXS/SANS), in combination with analytical centrifugation and light scattering, we have determined the solution properties of PFV IN alone and its synaptic complex with processed U5 viral DNA and related these properties to models derived from available crystal structures. PFV IN is a monomer in solution, and SAXS analysis indicates an ensemble of conformations that differ from that observed in the crystallographic DNA-bound state. Scattering data indicate that the PFV intasome adopts a shape in solution that is consistent with the tetrameric assembly inferred from crystallographic symmetry, and these properties are largely preserved in the presence of divalent ions and clinical strand transfer inhibitors. Using contrast variation methods, we have reconstructed the solution structure of the PFV intasome complex and have located the distal domains of IN that were unresolved by crystallography. These results provide important insights into the architecture of the retroviral intasome.

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A crystal structure of the catalytic core domain of an avian sarcoma and leukemia virus integrase suggests an alternate dimeric assembly.

Project description:Integrase (IN) is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV) inhibitors. This enzyme is composed of three domains and is hard to crystallize in its full form. First structural results on IN were obtained on the catalytic core domain (CCD) of the avian Rous and Sarcoma Virus strain Schmidt-Ruppin A (RSV-A) and on the CCD of HIV-1 IN. A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices (α1/α5, α5/α1). These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1) IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV-A by a single accessible residue in position 182 (substitution A182T). Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices (α3/α5, α1/α1, α5/α3). A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its α1, α3, α5 surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have subsequently determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV-A IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of retroviral INs, which beside integration of dsDNA are implicated in different steps of the retroviral cycle in presence of viral ssRNA.

| S-EPMC3153463 | biostudies-literature

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