Sialylatin of glycoproteins of murine mammary tumor virus, murine leukemia virus, and Mason-Pfizer monkey virus.

Neuraminidase treatment of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus resulted in loss of their capacity to inhibit hemagglutination of influenza virus. Hemagglutination-inhibition activity of these RNA tumor viruses could be restored by in vitro resialylation catalyzed by sialyl transferase. The major glycoprotein in the intact envelope of desialylated and, to some extent, native virions could be specificallly labeled in vitro with CMP-(14C) sialic acid. These studies further characterize the individual glycoproteins of mouse mammary tumor virus, Rauscher murine leukemia virus, and Mason-Pfizer monkey virus.     

Integrase of Mason-Pfizer monkey virus

The gene encoding an integrase of Mason-Pfizer monkey virus (M-PMV) is located at the 3'-end of the pol open reading frame. The M-PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M-PMV integrase and compared its activities and primary structure with those of HIV-1 and other retroviral integrases. M-PMV integrase prefers untranslated 3'-region-derived long-terminal repeat sequences in both the 3'-processing and the strand transfer activity assays. While the 3'-processing reaction catalyzed by M-PMV integrase was significantly increased in the presence of Mn(2+) and Co(2+) and was readily detectable in the presence of Mg(2+) and Ni(2+) cations, the strand transfer activity was strictly dependent only on Mn(2+). M-PMV integrase displays more relaxed substrate specificity than HIV-1 integrase, catalyzing the cleavage and the strand transfer of M-PMV and HIV-1 long-terminal repeat-derived substrates with similar efficiency. The structure-based sequence alignment of M-PMV, HIV-1, SIV, and ASV integrases predicted critical amino acids and motifs of M-PMV integrase for metal binding, interaction with nucleic acids, dimerization, protein structure maintenance and function, as well as for binding of human immunodeficiency virus type 1 and Rous avian sarcoma virus integrase inhibitors 5-CI-TEP, DHPTPB and Y-3.     

The betaretrovirus Mason-Pfizer monkey virus selectively excludes simian APOBEC3G from virion particles

The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroviruses. Therefore, any retrovirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Sch?fer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroviruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroviruses.     

RNA subunit structure of Mason-Pfizer monkey virus.

Mason-Pfizer monkey virus 60-70S RNA has a molecular weight of 8 times 10-6 when analyzed on polyacrylamide gels. Dissociation of 60-70S RNA of Mason-Pfizer monkey virus and murine leukemia virus by heat or formamide (40%) resulted in conversion to identical subunit structures of 2.8 times 10-6 daltons; treatment with lower amounts of formamide revealed a partial dissociation of Mason-Pfizer monkey virus 60-70S RNA released three low-molecular-weight RNA species of 10-5, 3,5 times 10-4, and 2.5 times 10-4.     

Effect of monensin on Mason-Pfizer monkey virus glycoprotein synthesis.

The effect of the monovalent carboxylic ionophore monensin on the biosynthesis, intracellular transport, and surface expression of the glycoproteins of Mason-Pfizer monkey virus was examined. Cells treated with monensin at concentrations of 10(-7) or 10(-6) M continued to synthesize virus particles, which from electron microscopic studies appeared to bud normally from the plasma membrane of the cells. However, the particles released had an altered buoyant density in sucrose gradients and were noninfectious. These noninfectious virions had a normal complement of non-glycosylated polypeptides but showed a significantly reduced amount of glycosylated proteins. The gp70 and gp20 polypeptides appeared to be completely absent, and a heterogeneous, higher-molecular-weight protein was observed on the virions instead. Studies on intracellular protein synthesis indicated that the precursor (Pr86env) to gp70 and gp20 is synthesized normally but is not cleaved to the mature proteins. Immunofluorescence studies showed, however, that the uncleaved molecule is expressed on the cell surface. In this system, therefore, Mason-Pfizer monkey virus glycoprotein migration appears to occur in the presence of monensin, whereas the cleavage and insertion of the glycoproteins into virions are inhibited.     

Secondary structure and mutational analysis of the Mason-Pfizer monkey virus RNA constitutive transport element.

The Mason-Pfizer monkey virus (MPMV) genome contains a cis-acting element that serves to facilitate nucleocytoplasmic export of intron-containing RNA. This element, known as the constitutive transport element (CTE), has been mapped to a 154-nt region close to the 3' end of the MPMV genome. The CTE contains a degenerate direct repeat of approximately 70 nt. We have probed the secondary structure of the CTE using double-strand- and single-strand-specific ribonucleases and chemical modification agents. A mutational analysis was also performed to confirm critical features of the CTE structure, as well as to identify regions that contain sequence-specific information required for function. Our results indicate that the CTE forms a long stem structure that contains a 9-nt terminal hairpin loop as well as two identical 16-nt inner loops. The inner loop sequences are rotated 180 degrees relative to each other within the structure. The mutational analysis shows that primary sequences in the loop regions are important for function, suggesting that they may contain binding sites for cellular proteins involved in RNA export. Interestingly, sequences with significant homology to the inner loop regions are found in the genomes of spumaviruses and mouse intracisternal A particles.     

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.     

Vaccinia virus A6 is essential for virion membrane biogenesis and localization of virion membrane proteins to sites of virion assembly

Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-β-D-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.     

Nucleotide sequence of Mason-Pfizer monkey virus: an immunosuppressive D-type retrovirus

The genetic structure of Mason-Pfizer monkey virus (MPMV), a D-type retrovirus, has been determined. In addition to the viral gag, pol, and env genes is an ORF overlapping both gag and pol and that encodes the viral protease. Surprisingly, the MPMV env protein is highly homologous to that of the avian C-type virus, reticuloendotheliosis associated virus REV-A. The env sequence encodes an immunosuppressive peptide, which suggests that MPMV, like REV-A, may transiently induce a T-suppressor cell population. The different phylogenies of the MPMV pol and env genes indicate a recombinatorial origin for the D-type viruses. Sequence comparisons show that SRV-1, an MPMV-like virus etiologically linked to simian AIDS (SAIDS), is in fact a variant of MPMV. While MPMV-like viruses cannot be used as direct models for the AIDS/SAIDS associated with lentiviruses, they provide an important system for studying the molecular basis of immunosuppressive diseases in primates.     

Importance of p12 protein in Mason-Pfizer monkey virus assembly and infectivity.

Mason-Pfizer monkey virus (M-PMV) represents the prototype type D retrovirus, characterized by the assembly of intracytoplasmic A-type particles within the infected-cell cytoplasm. These immature particles migrate to the plasma membrane, where they are released by budding. The gag gene of M-PMV encodes a novel protein, p12, just 5' of the major capsid protein (CA) p27 on the polyprotein precursor. The function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus and is absent from the type C retroviruses. In order to determine whether the p12 protein plays a role in the intracytoplasmic assembly of capsids, a series of in-frame deletion mutations were constructed in the p12 coding domain. The mutant gag genes were expressed by a recombinant vaccinia virus-T7 polymerase-based system in CV-1 cells or in the context of the viral genome in COS-1 cells. In both of these high-level expression systems, mutant Gag precursors were competent to assemble but were not infectious. In contrast, when stable transfectant HeLa cell lines were established, assembly of the mutant precursors into capsids was drastically reduced. Instead, the polyprotein precursors remained predominantly soluble in the cytoplasm. These results show that while p12 is not required for the intracytoplasmic assembly of M-PMV capsids, under the conditions of low-level protein biosynthesis seen in virus-infected cells, it may assist in the stable association of polyprotein precursors for capsid assembly. Moreover, the presence of the p12 coding domain is absolutely required for the infectivity of M-PMV virions.     

Coronavirus proteins: biogenesis of avian infectious bronchitis virus virion proteins.

We examined the synthesis of viral structural proteins in cultured cells infected with the avian coronavirus infectious bronchitis virus. Tryptic peptide mapping was used to determine the structural relationships of the intracellular proteins to the virion polypeptides. Pulse-chase experiments were performed to identify precursors to the virus-specific proteins. We found that the nucleocapsid protein, P51, and the small viral membrane proteins GP31, GP28, and P23 do not undergo post-translational proteolytic processing. In contrast, GP90 and GP84, the two large virion membrane proteins, were found to be produced by cleavage of a single precursor, GP155. This demonstrated that at least one coronavirus mRNA specifies two virion proteins.     

Effect of tunicamycin on cell fusion induced by Mason-Pfizer monkey virus.

Mason-Pfizer monkey virus, a D-type retrovirus, has been shown to induce multinucleate cell (syncytium) formation or cell fusion in several normal primate cells. A series of experiments has been carried out to examine whether a glycosylated "fusion-inducing" product is responsible for this biological property of Mason-Pfizer monkey virus. Treatment of rhesus monkey fetal lung cells with different concentrations of tunicamycin, a potent inhibitor of glycosylation, during infection with Mason-Pfizer monkey virus had no effect on cell fusion even though up to 5 micrograms of the drug per ml was tested. Furthermore, no significant effect on the extent of syncytium formation in rhesus monkey fetal lung cells was observed when the time of addition or duration of treatment with this inhibitor was varied. Nevertheless, tunicamycin was very effective in blocking glycosylation in rhesus cells since virions produced in the presence of this drug completely lacked gp70 and gp20, the two structural glycoproteins of Mason-Pfizer monkey virus. These non-glycosylated virus particles produced in the presence of tunicamycin were noninfectious as determined by a protein A binding assay and were unable to induce syncytium formation when assayed on rhesus cells. These results indicate that glycosylation of the fusion-inducing product is not required for multinucleate cell formation induced by Mason Pfizer monkey virus.     

Retrovirus D/New England and its relation to Mason-Pfizer monkey virus.

We have generated lymphocytic choriomeningitis virus-specific, H-2-restricted cytotoxic thymus-derived lymphocyte (CTL) clones. By using these reagents in several in vitro assays with infected target cells, we show that CTLs by themselves prevent the release of infectious virus into culture fluids and significantly lower the titers of infectious virus previously produced. This ability of cloned CTLs is not influenced by monensin. However, monensin does abrogate the ability of CTLs from spleens of mice primed 6 to 8 days previously with virus to kill virus-infected syngeneic targets. When tested for the participation of lymphokines in this system, the CTLs proliferate when reacted with syngeneic lymphocytic choriomeningitis virus-infected macrophages but fail to make interleukin-2. These CTLs make gamma interferon when reacted with syngeneic virus-infected targets. However, the production of interferon does not directly correlate with CTL-mediated killing. The number of H-2K and D molecules expressed on the target cell surface is not altered during the course of lymphocytic choriomeningitis virus infection. Electron microscopy shows finger-like projections of the CTL clone thrust into the infected cell and lesions bearing an internal diameter of approximately 15 nm in those membranes, illustrating the lytic process.     

Mutational analysis of the major homology region of Mason-Pfizer monkey virus by use of saturation mutagenesis.

The major capsid (CA) protein of retroviruses possesses a stretch of 20 amino acids, called the major homology region (MHR), which is evolutionarily conserved and invariant in location within the primary sequence of the protein. The function of this region was investigated by examining the effect of random single-amino-acid substitutions within the central 13 positions of the MHR on the life cycle of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive D-type retrovirus. When these mutants were subcloned into an M-PMV proviral vector and expressed in COS cells, one of two major phenotypes was observed. The first group, containing three mutants bearing drastic amino acid substitutions, was unable to assemble capsids in the cytoplasm of the host cell. The second and more common group of mutants was able to assemble and release virions, but these either displayed greatly reduced levels of infectivity or were completely noninfectious. Included within this second group were two mutants with unusual phenotypes; mutant D158Y exhibited a novel cleavage site for the viral protease that resulted in cleavage of the major capsid protein, p27 (CA), within the MHR, whereas mutant F156L appeared to have lost a major site for antibody recognition within the mature CA protein. The results of this mutagenic analysis suggest that changes in the MHR sequence can interfere with the assembly of viral capsids and block an early stage of the infection cycle of M-PMV.     

Distinct roles for nucleic acid in in vitro assembly of purified Mason-Pfizer monkey virus CANC proteins

In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (DeltaProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV DeltaProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which DeltaProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into DeltaProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.     

The Mason-Pfizer monkey virus PPPY and PSAP motifs both contribute to virus release

Late (L) domains are required for the efficient release of several groups of enveloped viruses. Three amino acid motifs have been shown to provide L-domain function, namely, PPXY, PT/SAP, or YPDL. The retrovirus Mason-Pfizer monkey virus (MPMV) carries closely spaced PPPY and PSAP motifs. Mutation of the PPPY motif results in a complete loss of virus release. Here, we show that the PSAP motif acts as an additional L domain and promotes the efficient release of MPMV but requires an intact PPPY motif to perform its function. Examination of HeLaP4 cells expressing PSAP mutant virus by electron microscopy revealed mostly late budding structures and chains of viruses accumulating at the cell surface with little free virus. In the case of the PPPY mutant virus, budding appeared to be mostly arrested at an earlier stage before induction of membrane curvature. The cellular protein TSG101, which interacts with the human immunodeficiency virus type 1 (HIV-1) PTAP L domain, was packaged into MPMV in a PSAP-dependent manner. Since TSG101 is crucial for HIV-1 release, this result suggests that the Gag-TSG101 interaction is responsible for the virus release function of the MPMV PSAP motif. Nedd4, which has been shown to interact with viral PPPY motifs, was also detected in MPMV particles, albeit at much lower levels. Consistent with a role of VPS4A in the budding of both PPPY and PTAP motif-containing viruses, the overexpression of ATPase-defective GFP-VPS4A fusion proteins blocked both wild-type and PSAP mutant virus release.     

dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency

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.     

Structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus.

We have found six major polypeptides in virions of the avian coronavirus infectious bronchitis virus grown in tissue culture: four glycoproteins, GP84, GP36, GP31, and GP28, and two non-glycosylated proteins, P51 and P23. In addition, we detected three minor species: two glycoproteins, GP90 and GP59, and one non-glycosylated protein, P14. Two-dimensional tryptic peptide mapping showed that GP36, GP31, GP28, and P23 comprise a group of closely related proteins which we have designated the "P23 family," but that the other proteins are distinct. Analysis by partial proteolytic digestion of P23 family, but that the other proteins are distinct. Analysis by partial proteolytic digestion of the P23 family labeled biosynthetically with [35S] methionine, and P23, labeled with [35S] formyl-methionine by in vitro translation of RNA from infected cells, revealed that the proteins of the P23 family differ in their amino-terminal domains. Similar analysis of GP31 and Gp36 labeled with [3H] mannose showed that the partial proteolytic fragments unique to these proteins were glycosylated. This suggests that differences in glycosylation in the amino-terminal domains contributes to the marked polymorphism os the P23 family. The results are discussed with respect to possible models for synthesis of the virion proteins.     

Identification of postentry restrictions to Mason-Pfizer monkey virus infection in New World monkey cells

TRIM5α has been shown to be a major postentry determinant of the host range for gammaretroviruses and lentiviruses and, more recently, spumaviruses. However, the restrictive potential of TRIM5α against other retroviruses has been largely unexplored. We sought to determine whether or not Mason-Pfizer monkey virus (M-PMV), a prototype betaretrovirus isolated from rhesus macaques, was sensitive to restriction by TRIM5α. Cell lines from both Old World and New World primate species were screened for their susceptibility to infection by vesicular stomatitis virus G protein pseudotyped M-PMV. All of the cell lines tested that were established from Old World primates were found to be susceptible to M-PMV infection. However, fibroblasts established from three New World monkey species specifically resisted infection by this virus. Exogenously expressing TRIM5α from either tamarin or squirrel monkeys in permissive cell lines resulted in a block to M-PMV infection. Restriction in the resistant cell line of spider monkey origin was determined to occur at a postentry stage. However, spider monkey TRIM5α expression in permissive cells failed to restrict M-PMV infection, and interference with endogenous TRIM5α in the spider monkey fibroblasts failed to relieve the block to infectivity. Our results demonstrate that TRIM5α specificity extends to betaretroviruses and suggest that New World monkeys have evolved additional mechanisms to restrict the infection of at least one primate betaretrovirus.