Recombinant human interferons inhibit replication of Mason-Pfizer monkey virus in primate cells

Pretreatment of Mason-Pfizer monkey virus-infected human embryonic kidney cells with either of the cloned human interferons, A or A/D, reduces the release of infectious virus particles to the same extent (greater than 97%) as determined by 125I-protein A binding radioimmune assay. While interferon A has no significant effect on the release of infectious viruses from a Mason-Pfizer monkey virus-infected monkey cell line consistent with the species specificity of this group of compounds, human interferon A/D can block the release of virions from the same monkey cell line. This block appears to be at the level of viral particle release from the cells since both labeling with radioactive precursors and electron microscopic observations show few extracellular particles but normal levels of intracytoplasmic A particles. The results show that cloned human interferon A/D can block the replication of an immunosuppressive retrovirus in primate cells and raise the possibility of testing the efficacy of this interferon in primate model systems.     

Commercial simian virus antisera that inhibit virus replication in primary monkey kidney cell cultures.

The incorporation of hyperimmune serum into cell culture medium to control endogenous viral infections of primary cells can have a significant effect on the replication of other viruses. When commercial simian virus 5 or simian virus 40 antiserum was used with primary monkey kidney cell cultures, we found a significant inhibition (greater than 90%) of the replication of parainfluenza virus types 2 and 3 and reovirus type 1. In the viral diagnostic laboratory, the use of hyperimmune serum with primary monkey kidney cells may result in failure to isolate certain viruses if these cells are not first washed free of hyperimmune serum.     

Characterization of Mason-Pfizer virus induced cell transformationin vitro

Summary Several types of cell strains and established cell lines of simian and human origin failed to demonstrate foci of altered cells following infection with the Mason-Pfizer monkey virus (M-PMV). However, most diploid cultures, after infection, lived longer and displayed the ability to grow in soft agar medium. The number of cell colonies developing in the soft agar was directly proportional to the amount of virus added to the culture. Two types of cell colonies were isolated from soft agar after infection of monkey foreskin cells with M-PMV. One had characteristic fibroblastic morphology, and the other showed an epithelioid cell phenotype. The ratio of fibroblastic colonies to epithelioid colonies was in excess of 20:1. The epithelioid cultures displayed a complete lack of topoinhibition, formed three dimensional cellular dome structures, and demonstrated significant karyotypic alterations. Fibroblastic sublines, on the other hand, did not show formation of domes but presented some lack of topoinhibition. The majority of cells in fibroblastic sublines also continued to show a normal rhesus chromosome complement. Although both epithelioid and fibroblastic transformed cell types produced intracellular M-PMV antigen and virus particles, the infectious virus titers were significantly different. The noninfectious virus preparations recovered from some of the fibroblastic sublines contained a high percentage of aberrant forms of M-PMV.With 6 Figures     

Detection of Mason-Pfizer monkey virus infection by syncytia formation of human cells doubly transformed by Rous sarcoma virus and simian virus 40

Summary Human cells doubly transformed by Rous sarcoma virus and SV40 (RSb cells) formed syncytia by cocultivation with Mason-Pfizer monkey virus (MPMV)-producing cells. This cell fusion was blocked by anti-MPMV serum indicating that the phenomenon is MPMV specific. The RSb cells were successfully used for MPMV infectivity assay in the same manner as KC cells.With 1 Figure     

The characterization of Mason-Pfizer monkey virus-induced cell fusion.

The characteristics and requirements of multinucleate cell (syncytium) induction by Mason-Pfizer monkey virus (M-PMV) on human and non-human primate cells have been investigated. Multinucleate cell induction by this “D”-type retrovirus shows single-hit kinetics on human foreskin and rhesus monkey fetal lung cells. The peak of syncytium-forming activity in an isopycnic sucrose gradient coincides with the peak of M-PMV virions as assessed by electron microscopy and analysis of viral polypeptides. Unlike the paramyxoviruses, M-PMV does not induce “early” cell fusion when added in high concentrations to the size of the syncytia remains constant after this time. Ultraviolet irradiation of M-PMV reduces its ability to form syncytia and to replicate with single-hit kinetics, suggesting that a functional viral genome is required for syncytium formation. Proviral DNA synthesis and assembly of virions are not necessary for cell fusion since the addition of cytosine arabinoside at concentrations which block virus replication has little effect on multinucleate cell formation. Moreover both multinucleate cells lacking detectable intracellular virus polypeptides, and groups of individual, nonfused but brightly staining cells can be observed in immunofluorescence assays at times when multinucleate cell formation is maximal. Cell fusion is inhibited by the addition of cycloheximide during the first 12 hr of infection, suggesting that de novo protein synthesis is required for multinucleate cell formation. The possibility that translation of genomic RNA yields a fusion-inducing product is discussed.     

Detection of simian type-C particles in Mason-Pfizer virus infected cultures.

A simian type-C virus has been detected in cultures chronically infected with Mason-Pfizer monkey virus (M-PMV). Simultaneous budding of M-PMV and type-C virus particles from the same cells was observed in cultures incubated at 37 or 40 degrees C. However, the frequency of such cells was greater in cultures grown at 40 degrees C. Although clusters of type-C viral buds were seen at the surface of the cells, extracellular mature type-C particles in cell pellets or concentrated virus preparations were very rarely found. The increase in frequency of type-C buds was found to be transitory since cultures adapted to growing at the high temperature demonstrated budding type-C particles only occasionally. Cultures producing type-C buds were found to contain, in addition to M-PMV antigens, serological activity with polyvalent antisera produced against multiple structural components of endogenous baboon virus (BV) or simian sarcoma virus (SiSV). The reactivity, however, was found not to be serologically related to the major SiSV P28 core protein.     

Atomic force microscopy investigation of Mason-Pfizer monkey virus and human immunodeficiency virus type 1 reassembled particles

Particles of DeltaProCANC, a fusion of capsid (CA) and nucleocapsid (NC) protein of Mason-Pfizer monkey virus (M-PMV), which lacks the amino terminal proline, were reassembled in vitro and visualized by atomic force microscopy (AFM). The particles, of 83-84 nm diameter, exhibited ordered domains based on trigonal arrays of prominent rings with center to center distances of 8.7 nm. Imperfect closure of the lattice on the spherical surface was affected by formation of discontinuities. The lattice is consistent only with plane group p3 where one molecule is shared between contiguous rings. There are no pentameric clusters nor evidence that the particles are icosahedral. Tubular structures were also reassembled, in vitro, from two HIV fusion proteins, DeltaProCANC and CANC. The tubes were uniform in diameter, 40 nm, but varied in length to a maximum of 600 nm. They exhibited left handed helical symmetry based on a p6 hexagonal net. The organization of HIV fusion proteins in the tubes is significantly different than for the protein units in the particles of M-PMV DeltaProCANC.     

Morphological observations on the replication of herpesvirus saimiri in monkey kidney cell cultures.

Owl and African green monkey kidney cell cultures have been infected with 1 p.f.u./cell of herpesvirus saimiri and sample cultures have been taken for examination by electron microscopy at 3 to 6 hourly intervals over a period of 7 days; the experiments were repeated several times. The peculiarly slow replication cycle of Herpesvirus saimiri has enabled distinct cytoplasmic and nuclear phases in virus maturation to be clearly distinguished; the overall fine structural features were similar in both cell types. Immature particles were first detected in the nucleus and cytoplasm 63 h after infection. Thereafter, abundant cytoplasmic immature particles matured by budding through cytoplasmic membranes until about 100 h, whereas nuclear immature particles budded through the inner nuclear membrane or intranuclear invaginations of it later, from about 100 h until cytolysis was complete at 160 h. Morphological differences were also observed between particles budding at cytoplasmic membranes and the nuclear envelope. At the former site the membrane overlying the bud showed an electron opaque thickening which imparted to the mature particle an asymmetrical appearance. Such thickenings of the envelope were not observed in mature particles of nuclear origin. Unusual tubular and laminated nuclear structures were seen towards the end of the replicative cycle corresponding with the phase of nuclear virus maturation by budding; the morphology of the latter structures is described.     

Isolation and characterization of the Mason-Pfizer monkey virus p12 protein

The Mason-Pfizer monkey virus (M-PMV) Gag protein, precursor to the structural proteins of the infectious virion, assembles into immature capsid-like particles when expressed at high levels in bacterial cells. Similar capsid-like particles can be obtained by in vitro assembly using a high concentration of isolated Gag. M-PMV Gag contains a p12 protein that has no corresponding analogues in most other retroviruses and has been suggested to contain an internal scaffold domain (ISD). We have expressed and purified p12 and the N- and C-terminal halves (Np12 and Cp12) that are predicted to be structurally independent domains. The behavior of these proteins was analyzed using chemical cross-linking, CD spectroscopy, and electron microscopy. The N-terminal half of p12 is largely alpha-helical although the C-terminal portion lacks any apparent ordered structure. Both p12 and Np12 form high-order oligomers in vitro and when expressed in E. coli produce organized structures that are visible by electron microscopy. Interestingly, Cp12, as well as the whole protein, can form dimers in the presence of SDS. The data show that both domains of p12 contribute to its ability to multimerize with much of this potential residing in its N-terminal part, most probably within the leucine zipper-like (LZL) sequence.     

Bovine parainfluenza type 3 virus infection: virus replication in bovine embryonic cell cultures and virion separation by rate-zonal centrifugation.

Replicative sequences of a bovine strain of parainfluenza type 3 virus in bovine embryonic kidney and spleen cell cultures were investigated by light and fluorescence microscopy and by ultrathin section and negative-contrast electron microscopy. Observations from light and fluorescence microscopy showed that intracytoplasmic inclusions were detected as small granules surrounding the nuclei of more than 90 percent of the cell population by day 2 postinoculation. With the increase of postexposure times, these inclusions coalesced into larger bodies which occupied large portions of the cell. Ultrastructurally, the first sign of virus development was the appearance of aggregates of viral nucleocapsids in the vicinity of the nucleus. With the concomitant accumulation of viral nucleocapsids in the cytoplasm, the virus maturation was expressed by budding processes through the cell membrane into round, oval, or elongated forms. Eosinophilic inclusions were demonstrable in many mitotic cells. Ultrastructurally, these cells were observed to produce virus particles by a process identical to that of resting cells. Virions, prepared from infected culture fluid and negatively stained, appeared to be pleomorphic and their diameter ranged from 200 to 600 mm. The virions were separated, by rate-zonal centrifugation, into two subclasses in a sucrose gradient (15 to 60 percent, wt/wt). The slowly sedimenting virions had a density approximately 1.20 gm/cm3 and an average size of 200 nm in diameter, whereas the faster-sedimenting virions had a density of 1.24 gm/cm3 and average diameter of 400 nm.     

Serological analysis of reverse transcriptase of the Mason-Pfizer monkey virus

Antiserum prepared against partially purified DNA polymerase from Mason-Pfizer monkey virus neutralized the endogenous DNA polymerase of that virus and of X381, an agent morphologically indistinguishable from MPMV, isolated from cultures prepared from the lactating mammary gland of a rhesus monkey. The antiserum did not inhibit the DNA polymerase activities of avian myeloblastosis virus, feline leukemia virus, Rauscher murine leukemia virus, Friend murine leukemia virus, murine mammary tumor virus, and simian sarcoma virus-1.     

Persistent SV5 virus infection in continuous cell cultures.

A continuous line of guinea pig kidney cells (CGPK/H) and a continuous line of mouse fibroblasts (L/H) spontaneously infected with parainfluenza virus SV5 were found. These cultures showed no enhanced cell degeneration or symplast formation, nor was haemagglutinin accumulation or infectious virus demonstrated in them. However, regular reproduction of ribonucleoprotein (RNP) characteristic of parainfluenza viruses, morphologically complete virions and antigens producing antibody to SV5 virus were found in the cells. Focal haemadsorption neutralized by antiserum to SV5 virus was also demonstrated. The infection persisted in the cell populations for over 2 years (the observation period) under standard conditions of cell dispersion and subcultivation.     

Mason-Pfizer monkey virus Gag proteins interact with the human sumo conjugating enzyme, hUbc9

Retroviral Gag proteins function during early and late stages of the viral life cycle. To gain additional insight into the cellular requirements for viral replication, a two-hybrid screen was used to identify cellular proteins that interact with the Mason-Pfizer monkey virus Gag protein. One of the cellular proteins found was identified as hUbc9, a nuclear pore-associated E2 SUMO conjugating enzyme. In vitro protein interaction assays verified the association and mapped the interaction domain to the CA protein. In vivo, hUbc9 and Gag colocalized in the cytoplasm as discrete foci near the nuclear membrane. In addition, overexpression of hUbc9 in cells caused a fraction of Gag to colocalize with hUbc9 in the nucleus. These experiments demonstrate that hUbc9 and Gag interact in cells, strengthen the hypothesis that Gag proteins transiently associate with the nuclear compartment during viral replication, and suggest that hUbc9 plays a role in this process.     

Squirrel monkey retrovirus: Electron microscopy of a virus from new world monkeys and comparison with Mason-Pfizer monkey virus

Summary The ultrastructural morphogenesis of squirrel monkey retrovirus (SMRV) and Mason-Pfizer monkey virus (MPMV) grown in cell culture were compared. Both viruses develop by a process that begins with the formation of intracytoplasmic A particles which are then enveloped at the plasma membrane during budding. SMRV also develops as a crescent-shaped nucleoid beneath a bulging plasma membrane, a development characteristic of type C oncornaviruses. Free extracellular mature SMRV was generally round with a centrally located electron-dense nucleoid enclosed by the viral envelope. In contrast, mature MPMV had a tubular-shaped nucleoid. Negative stained preparations of both viruses yielded head-tail forms with surface projections. By uranyl acetate/critical point drying, SMRV particles were usually round with an eccentric electron-dense nucleoid enclosed by the viral envelope, whereas MPMV particles were round and contained an electron-dense bar-shaped nucleoid. These morphological observations indicate that SMRV more closely resembles MPMV, presently the only member of genus oncornavirus type D, than other retroviruses species. However, since SMRV can be morphologically, biochemically, and immunologically distinguished from MPMV, it represents a new species within genus oncornavirus type D.With 5 Figures     

Dexamethasone enhances human cytomegalovirus replication in human epithelial cell cultures

An epithelial human hepatoma cell line (PLC/PRF/5) and a primary epithelial human baby kidney (HBK) cell culture showed restricted growth of human cytomegalovirus (HCMV). Treatment of these two epithelial cell cultures with dexamethasone greatly enhanced their ability to support HCMV replication. Growth kinetic experiments and infectious center assay revealed that in both the hormone-treated cultures infectious progeny virus appeared earlier by 1 or 2 days and 5- or 10-fold more cells are able to produce infectious virus. There was an approximate 50- or 100-fold increase in virus yield compared to that in the untreated control cultures. Enhanced HCMV replication in the hormone-treated cultures was not due to differences in the cell growth or the virus adsorption and was supported by evidence of increased synthesis of HCMV-specific immediate early antigens and DNA.     

Respiratory syncytial virus infection of human primary nasal and bronchial epithelial cell cultures and bronchoalveolar macrophages.

In adults, clinical symptoms caused by respiratory syncytial virus (RSV) are usually confined to the upper respiratory tract, whereas RSV infection in infants frequently causes bronchiolitis and pneumonia. The preferential localization of RSV infection to the upper airways may partially be due to protective immunity, but may also depend on a difference in susceptibility of epithelial cells from upper and lower airways, or on antiviral activities of bronchoalveolar macrophages (AM). In this study, we have compared the susceptibility of primary adult human nasal epithelium, primary adult human bronchial epithelium, a human bronchial epithelial cell line (BEAS-2B), and adult human AM to infection with RSV. The cell cultures were infected with multiplicities of infection (moi) of 1 and 0.1. Virus release into the supernatants was assayed at days 1, 2, 4, and 7, and the percentage of virus-positive cells determined by immunofluorescence at the same time points. Similar proportions of nasal epithelial cells (NE) and bronchial epithelial cells (BE) were infected with RSV. Approximately 50 to 75% (with moi 1) and 2 to 10% (with moi 0.1) of the cells were infected by 24 h; almost all the cells were RSV positive by day 4. However, BE released less infectious RSV than do NE. With moi 0.1, 10-fold less virus was released over 4 days of culture. By days 4 to 7, cytopathic effects (CPE) were maximal in all epithelial cell cultures, but CPE developed latest in BE infected with moi 0.1. AM were also productively infected with RSV, with peak virus production at day 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Type D retroviruses: occurrence of natural antibodies to Mason-Pfizer monkey virus in rhesus monkeys.

Sera from rhesus monkeys maintained at three separate geographic regions were found to possess naturally occurring antibody to the Mason-Pfizer monkey virus (MPMV) major structural protein. The specificity of this reactivity was established by absorption with purified viral protein. While animals from two of these colonies were housed in association with MPMV-infected monkeys, no such prior exposure to virus-infected animals could be traced in the case of the third colony. These results in combination with the previous inability to detect full complements of MPMV genetic sequences in the cellular DNA of the normal rhesus monkeys provide evidence for horizontal transmission of MPMV. Alternatively, the present findings could reflect humoral immune response to expression of an endogenous type D retrovirus highly related to MPMV.     

Differentiation of type D virus from transplantable human cells (Il'in-Bykovskii virus) and Mason-Pfizer monkey virus on the basis of virus envelope antigens

To differentiate between Il'in-Bykovskii virus (IBV) and Mason-Pfizer monkey virus (MPMV) the method of neutralization of the viruses by antisera against virus envelope antigens was used. The viruses were cultured on the same human embryonic cells. The results of virus neutralization were determined by the presence or absence of gs antigen in the affected cells. Antiserum against IBV envelope antigens neutralized IBV but not MPMV. Antiserum against MPMV did not neutralize IBV. It is concluded that IBV and MPMV differ in their virus envelope antigens and must be regarded as different viruses.Laboratory of Sarcomoleukemic Viruses, Department of Etiology and Immunology of Tumors, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 8, pp. 208–210, August, 1977.     

Frog virus 3 replication: electron microscope observations on the terminal stages of infection in chronically infected cell cultures.

An examination of BHK, CEF, and FHM cells chronically infected with frog virus 3 has been made by scanning and transmission (thin section, freeze fracture, and surface replica) electron microscopy. With minor differences the pattern of virus development is similar in all three cell line. Virus particles were detected in cell nuclei which subsequently became degenerate very late in infection. Three inclusions were associated with frog virus 3 cytoplasmic foci of infection; lamella structures, extensive microtubule formation (in BHK and FHM cells), and linear crystalline structures. The last two structures may play a role in creating or maintaining the cell rounding c.p.e. revealed by scanning electron microscopy. Very late in infection most BHK and FHM, but not CEF, cells are stripped of the plasma membrane. Replicas of frozen fractured BHK cells featured cytoplasmic foci of infection, budding at the plasma membrane, and showed that at early times when virus is detected in the nucleus, the nuclear membranes are intact and morphologically unaltered. Budding at the plasma membrane was better resolved by scanning and as surface replicas. This demonstrated that sparse to profuse localized budding occurred. Frequently virus particles were located singly, or as multiples, at the end of, or along, cytoplasmic protrusions which occur both on the body of the cells and at the cytoplasmic/coverslip 'interface'.