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ABSTRACT: Mason-Pfizer monkey virus (M-PMV), like some other betaretroviruses, encodes a G-patch domain (GPD). This glycine-rich domain, which has been predicted to be an RNA binding module, is invariably localized at the 3' end of the pro gene upstream of the pro-pol ribosomal frameshift sequence of genomic RNAs of betaretroviruses. Following two ribosomal frameshift events and the translation of viral mRNA, the GPD is present in both Gag-Pro and Gag-Pro-Pol polyproteins. During the maturation of the Gag-Pro polyprotein, the GPD transiently remains a C-terminal part of the protease (PR), from which it is then detached by PR itself. The destiny of the Gag-Pro-Pol-encoded GPD remains to be determined. The function of the GPD in the retroviral life cycle is unknown. To elucidate the role of the GPD in the M-PMV replication cycle, alanine-scanning mutational analysis of its most highly conserved residues was performed. A series of individual mutations as well as the deletion of the entire GPD had no effect on M-PMV assembly, polyprotein processing, and RNA incorporation. However, a reduction of the reverse transcriptase (RT) activity, resulting in a drop in M-PMV infectivity, was determined for all GPD mutants. Immunoprecipitation experiments suggested that the GPD is a part of RT and participates in its function. These data indicate that the M-PMV GPD functions as a part of reverse transcriptase rather than protease.
Journal of Virology 12/2011; 86(4):1988-98. · 5.40 Impact Factor
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ABSTRACT: Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus of CA to fold back to form a β-hairpin, anchored by an internal salt bridge between the N-terminal proline and the inner aspartate. Using an in vitro assembly system of capsid-nucleocapsid protein (CANC), we studied the formation of virus-like particles (VLP) of a gammaretrovirus, the xenotropic murine leukemia virus (MLV)-related virus (XMRV). We show here that, unlike other retroviruses, XMRV CA and CANC do not assemble tubular particles characteristic of mature assembly. The prevention of β-hairpin formation by the deletion of either the N-terminal proline or 10 initial amino acids enabled the assembly of ΔProCANC or Δ10CANC into immature-like spherical particles. Detailed three-dimensional (3D) structural analysis of these particles revealed that below a disordered N-terminal CA layer, the C terminus of CA assembles a typical immature lattice, which is linked by rod-like densities with the RNP.
Journal of Virology 11/2011; 86(3):1297-306. · 5.40 Impact Factor
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ABSTRACT: Acyclic nucleotide analogue PMEG represents promising drug candidate against lymphomas. In the present work we describe the ability of PMEG to induce resistance and we elucidate the mechanisms involved in this process. CCRF-CEM T-lymphoblastic cells resistant to either PMEG or its 6-amino congener PMEDAP were prepared and assayed for the expression of membrane transporters, PMEG and PMEDAP uptake and intracellular metabolism. Genes for guanylate kinase (GUK) and adenylate kinase (AK) isolated from PMEG- and PMEDAP-resistant cells were sequenced and cloned into mammalian expression vectors. PMEG-resistant cells were transfected with GUK vectors and catalytic activities of GUKs isolated from PMEG-sensitive and resistant cells were compared. PMEG phosphorylation to PMEG mono- and diphosphate was completely impaired in resistant cells. GUK obtained from PMEG-resistant cells revealed two point mutations S(35)N V(168)F that significantly suppressed its catalytic activity. Transfection of resistant cells with wtGUK led to the recovery of phosphorylating activity as well as sensitivity towards PMEG cytotoxicity. No differences in PMEG uptake have been found between sensitive and resistant cells. In contrast to GUK no changes in primary sequence of AK isolated from PMEDAP resistant cells were identified. Therefore, resistance induced by PMEDAP appears to be conferred by other mechanisms. In conclusion, we have identified GUK as the sole molecular target for the development of acquired resistance to the cytotoxic nucleotide PMEG. Therefore, PMEG is unlikely to cause cross-resistance in combination therapeutic protocols with most other commonly used anticancer drugs.
Biochemical pharmacology 07/2011; 82(2):131-8. · 4.25 Impact Factor
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ABSTRACT: Assembly of immature retroviral particles is a complex process involving interactions of several specific domains of the Gag polyprotein localized mainly within capsid protein (CA), spacer peptide (SP), and nucleocapsid protein (NC). In the present work we focus on the contribution of NC to the oligomerization of CA leading to assembly of Mason-Pfizer monkey virus (M-PMV) and HIV-1. Analyzing in vitro assembly of substitution and deletion mutants of DeltaProCANC, we identified a "spacer-like" sequence (NC(15)) at the M-PMV NC N terminus. This NC(15) domain is indispensable for the assembly and cannot be replaced with oligomerization domains of GCN4 or CREB proteins. Although the M-PMV NC(15) occupies a position analogous to that of the HIV-1 spacer peptide, it could not be replaced by the latter one. To induce the assembly, both M-PMV NC(15) and HIV-1 SP1 must be followed by a short peptide that is rich in basic residues. This region either can be specific, i.e., derived from the downstream NC sequence, or can be a nonspecific positively charged peptide. However, it cannot be replaced by heterologous interaction domains either from GCN4 or from CREB. In summary, we report here a novel M-PMV spacer-like domain that is functionally similar to other retroviral spacer peptides and contributes to the assembly of immature-virus-like particles.
Journal of Virology 12/2009; 84(4):1977-88. · 5.40 Impact Factor
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Pavel Macek,
Josef Chmelík,
Ivana Krízová,
Pavel Kaderávek,
Petr Padrta,
Lukás Zídek,
Marcela Wildová,
Romana Hadravová,
Radka Chaloupková,
Iva Pichová,
Tomás Ruml, Michaela Rumlová,
Vladimír Sklenár
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ABSTRACT: The high-resolution structure of the N-terminal domain (NTD) of the retroviral capsid protein (CA) of Mason-Pfizer monkey virus (M-PMV), a member of the betaretrovirus family, has been determined by NMR. The M-PMV NTD CA structure is similar to the other retroviral capsid structures and is characterized by a six alpha-helix bundle and an N-terminal beta-hairpin, stabilized by an interaction of highly conserved residues, Pro1 and Asp57. Since the role of the beta-hairpin has been shown to be critical for formation of infectious viral core, we also investigated the functional role of M-PMV beta-hairpin in two mutants (i.e., DeltaP1NTDCA and D57ANTDCA) where the salt bridge stabilizing the wild-type structure was disrupted. NMR data obtained for these mutants were compared with those obtained for the wild type. The main structural changes were observed within the beta-hairpin structure; within helices 2, 3, and 5; and in the loop connecting helices 2 and 3. This observation is supported by biochemical data showing different cleavage patterns of the wild-type and the mutated capsid-nucleocapsid fusion protein (CANC) by M-PMV protease. Despite these structural changes, the mutants with disrupted salt bridge are still able to assemble into immature, spherical particles. This confirms that the mutual interaction and topology within the beta-hairpin and helix 3 might correlate with the changes in interaction between immature and mature lattices.
Journal of Molecular Biology 07/2009; 392(1):100-14. · 4.00 Impact Factor
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ABSTRACT: Most retroviruses employ a frameshift mechanism during polyprotein synthesis to balance appropriate ratios of structural proteins and enzymes. To investigate the requirements for individual precursors in retrovirus assembly, we modified the polyprotein repertoire of Mason-Pfizer monkey virus (M-PMV) by mutating the frameshift sites to imitate the polyprotein organization of Rous sarcoma virus (Gag-Pro and Gag-Pro-Pol) or Human immunodeficiency virus (Gag and Gag-Pro-Pol). For the "Rous-like" virus, assembly was impaired with no incorporation of Gag-Pro-Pol into particles and for the "HIV-like" virus an altered morphogenesis was observed. A mutant expressing Gag and Gag-Pro polyproteins and lacking Gag-Pro-Pol assembled intracellular particles at a level similar to the wild-type. Gag-Pro-Pol polyprotein alone neither formed immature particles nor processed the precursor. All the mutants were non-infectious except the "HIV-like", which retained fractional infectivity.
Virology 01/2009; 384(1):59-68. · 3.35 Impact Factor
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ABSTRACT: Retroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroviruses refold after proteolytic cleavage into a beta-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer monkey virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50-Asp57 plays an important role in stabilization of the beta-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA.
Virology 09/2008; 380(1):157-63. · 3.35 Impact Factor
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Jirí Vlach,
Jan Lipov, Michaela Rumlová,
Václav Veverka,
Jan Lang,
Pavel Srb,
Zdenek Knejzlík,
Iva Pichová,
Eric Hunter,
Richard Hrabal,
Tomás Ruml
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ABSTRACT: Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason-Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.
Proceedings of the National Academy of Sciences 07/2008; 105(30):10565-70. · 9.68 Impact Factor
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ABSTRACT: Mason-Pfizer monkey virus (M-PMV) belongs to the family of betaretroviruses characterized by the assembly of immature particles within cytoplasm of infected cells in contrast to other retroviruses (e.g. HIV, RSV) that assemble their immature particles at a plasma membrane. Simultaneously with or shortly after budding a virus-encoded protease is activated and the Gag polyprotein is cleaved into three major structural proteins: matrix (MA), capsid (CA), and nucleocapsid (NC) protein. Mature retroviral CA proteins consist of two independently folded structural domains: N-terminal domain (NTD) and C-terminal dimerization domain (CTD), separated by a flexible linker. As a first step toward the solution structure elucidation, we present nearly complete backbone and side-chain 1H, 13C and 15N resonance assignment of the M-PMV NTD CA.
Biomolecular NMR Assignments 07/2008; 2(1):43-5. · 0.72 Impact Factor
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ABSTRACT: We have developed a sensitive luminometric assay for determining the activity of retroviral proteases that uses proteolytic cleavage of polypeptide substrate immobilized on Ni-NTA HisSorb Strips microplates. The protease substrate derived from the Gag precursor protein of Mason-Pfizer monkey virus (M-PMV) was conjugated with horseradish peroxidase (HRP), which catalyzes oxidation of luminol in the assay. The cleavage of the substrate was monitored as a decrease in luminescent signal caused by the release of the cleavage product conjugated to HRP. Testing of a set of M-PMV protease inhibitors confirmed that this method is sufficiently sensitive and specific for high-throughput screening of retroviral protease inhibitors.
Analytical Biochemistry 11/2005; 345(1):96-101. · 3.00 Impact Factor
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ABSTRACT: Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro. We show that recombinant betaretroviral NC-dUTPase and dUTPase are both inefficient catalysts compared with all other dUTPases. Dynamic light scattering and gel filtration confirm that the homotrimeric organization, common among dUTPases, is retained in the NC-dUTPase fusion protein. The betaretroviral dUTPase has been crystallized and single crystals contain homotrimers. Oligonucleotide and Zn2+ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase. Binding of the hexanucleotide ACTGCC or the octanucleotide (TG)4 to NC-dUTPase modulates enzymatic function, indicating that the low catalytic activity may be compensated by adequate localization.
Journal of Biological Chemistry 11/2003; 278(40):38803-12. · 4.77 Impact Factor
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ABSTRACT: Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration.
Virology 07/2003; 310(2):310-8. · 3.35 Impact Factor
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