The errors in assembly of MuLV in interferon treated cells

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine-containing, fucose-deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71-p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon-treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon-treated and -untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.     

Glycoprotein enrichment in Moloney leukemia virus structural proteins released from interferon-treated cells

Interferon treatment of Moloney-leukemia-virus-infected cells (3T3/MLV) leads to the formation of virus particles enriched with viral structural glycoproteins, in addition to the inhibition of virus production. A preferential inhibitory effect on incorporation of RNA and proteins rather than glycoproteins was found in the released virus particles from interferon-treated cells. Enrichment in 70,000- and 45,000-dalton glycoprotein (gP-70, gP-45) in these particles was further demonstrated by polyacrylamide analysis of viral proteins pulse-labeled with [3H]-leucine. Viral glycoproteins released as soluble antigens were also determined. A 40% reduction was found in gP-70 and gP-45 released from interferon-treated cells. Radioimmunoprecipitation of pulse-chase-labeled cellular viral proteins showed no effect of interferon on the formation of viral structural 30,000-, 15,000- to 12,000-dalton proteins, and gP-70 and gP-45 from their respective precursors. The uncoordinate effect of interferon inhibition on viral 30,000-dalton protein and gP-70 is discussed.     

Presence of murine leukemia virus envelope proteins gp70 and p15(E) in a common polyprotein of infected cells.

The murine leukemia virus envelope proteins, p15(E) and gp70, exhibit a mode of processing distinct from that of virion core proteins according to three criteria. First, the incorporation of both p15(E) and gp70 into virions is more sensitive to the metabolic analogue 2-deoxy-D-glucose than the incorporation of core proteins. Second, the kinetics with which the newly synthesized envelope proteins appear in the released virions is delayed relative to the appearance of core proteins. Third, immunoprecipitation of large polypeptides from infected cells reveals the presence of gp70 and p15(E) in a common precursor distinct from the core polyprotein.     

Effects of monensin on morphogenesis and infectivity of Friend murine leukemia virus.

The transport of the gp70 glycoprotein to the cell surface and concomitant release of infectious virus was inhibited by treatment of Friend murine leukemia virus-infected Eveline cells with the sodium ionophore monensin. Virus yields were reduced more than 50-fold by 10(-5) M monensin, whereas particle production was reduced by 50% in monensin-treated cells. The resulting particles failed to incorporate newly synthesized gp70 and p15(E), whereas the other structural proteins, p30, p15, p12, and p10, were incorporated into virions. However, monensin did not inhibit the incorporation into virions of preformed gp70. A reduction in the efficiency of cleavage of the PrENV glycoprotein precursor and a defect in the processing of simple endo-H-sensitive to complex endo-H-resistant oligosaccharides suggest that intracellular transport of gp70 may be blocked before its entry into the Golgi apparatus. Fewer particles were found to bud from the cell surface, but intracellular vacuoles with budding virions were detected. Ferritin labeling and pulse-chase studies suggested a cell surface origin for these vacuoles. These experiments indicate that monensin inhibits the transport of Friend murine leukemia virus glycoproteins at an early stage, with a resultant block in the assembly and release of infectious virus.     

A murine leukemia virus mutant with a temperature-sensitive defect in membrane glycoprotein synthesis.

Cells infected with a temperature-sensitive mutant (ts-26) of Rauscher murine leukemia virus (R-MuLV) or with wild-type virus were labeled with 35S-methionine, and cell extracts were examined for radioactive polypeptides which could be precipitated by monospecific antisera to viral proteins. When shifted from permissive (31 degrees C) to nonpermissive (39 degrees C) temperature, cells infected with ts-26 rapidly begin to accumulate gPr90enr, the glycoprotein precursor to the membrane envelope glycoprotein gp70 and to the membrane-associated protein p15E. Simultaneously, formation of these mature virion proteins ceases. In addition, lactoperoxidase-catalyzed surface labeling with 125I--iodine indicates that the plasma membrane of cells infected with ts-26 becomes depleted of gp70 antigens at 39 degrees C. Nevertheless, at 39 degrees C these cells release defective MuLVs which lack gp70 and p15E but contain an outer membrane. The released particles also contain an aberrantly processed form of the major virion core protein p30, and many of these virion cores have an unusual immature crescent shape. It has previously been reported that cells infected with the ts-26 mutant of R-MuLV process a 65,000 dalton precursor (Pr65gag) of the virion core proteins more slowly at 39 degrees C than do cells infected with wild-type virus (Stephenson, Tronick and Aaronson, 1975). Although we have confirmed these results, this effect is relatively small and it is known that various alterations of MuLV assembly can lead secondarily to inhibited processing of Pr65gag. We propose that the ts-26 mutant has a primary temperature-sensitive defect in membrane glycoprotein synthesis and that this change causes pleiotropic effects on core morphogenesis.     

Alterations of a mouse mammary tumor virus glycoprotein with interferon treatment.

The effects of exogenous mouse interferon on the MJY-alpha mammary tumor cell line chronically infected with mouse mammary tumor virus (MMTV) were examined. Interferon at concentrations of 25 to 2,000 IU/ml in culture medium did not alter the growth rate or morphology of the cell layers. Electron microscopic examination of interferon-treated cells indicated a decrease in the numbers of A-type and budding B-type particles of MMTV. However, the levels of extracellular MMTV virions in the culture supernatants were not significantly reduced. Profiles of MMTV glycoproteins and nonglycosylated polypeptides obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from interferon-treated cultures revealed increases in the relative levels of the 60,000-dalton glycoprotein, gp60.     

Inhibition of Rous sarcoma virus replication by 2-deoxyglucose and tunicamycin: identification of an unglycosylated env gene product.

Two inhibitors of glycosylation, 2-deoxyglucose and tunicamycin, depressed the synthesis of infectious Rous sarcoma virus greater than 100-fold. Under the same conditions only a two- to threefold decrease in the production of virus particles was observed. The noninfectious particles had a lower density (1.145 g/ml) in isopycnic sucrose gradients and lacked the two virion glycoproteins, gp85 and gp37, found on infectious virions. The four internal structural proteins of the virus, p27, p19, p15, and p12, appeared to be assembled normally into the noninfectious virus. Polypeptides related to the Rous sarcoma virus glycoproteins were immunoprecipitated from pulse-labeled Rous sarcoma virus (Prague strain, subgroup B)-transformed cells. pr95gp, the polyprotein precursor to gp85 and gp37, was the major protein precipitated from untreated cells. PR95GP, THE POLYPROTEIN PRECURSOR TO GP85 AND GP37, WAS THE MAJOR PROTEIN PRECIPITATED FROM UNTREATED CELLS. This was absent in both tunicamycin- and 2-deoxyglucose-treatec ells, and a new polypeptide of molecular weight 57,000 to 58,000 was the major species precipitated. In tunicamycin-treated cells this product was unstable and was degraded during a 2-h chase; in 2-deoxyglucose-treated cells, on the other hand, the polypeptide appeared to be more stable and underwent partial glycosylation. The synthesis and processing of pr76, the polyprotein precursor to the internal structural proteins of the virion, occurred normally in both treated and untreated cells. It is concluded that the unglycosylated env gene product is a polypeptide of molecular weight 57,000 to 58,000.     

Viral protein synthesis in mouse hepatitis virus strain A59-infected cells: effect of tunicamycin.

We identified eight protein species in virions of mouse hepatitis virus strain A59. Based on their sizes, prosthetic groups, and locations in virions, these proteins were designated gp180/E2, gp90/E2, pp54/N, gp26.5/E1, gp25.5/E1, p24/E1, p22/X, and p14.5/Y. The positions of the last two proteins in virions are not known. Host protein synthesis in Sac(-) cells infected with mouse hepatitis virus strain A59 was inhibited, and the following novel proteins appeared: gp150, gp90, p54, gp26.5, gp25.5, p24, p22, and p14.5. Except for gp150, these polypeptides all co-electrophoresed with mouse hepatitis virus strain A59 structural proteins. In addition, all of these proteins could be immunoprecipitated with a convalescent mouse serum or a rabbit antiserum raised against purified disrupted virus. After a 15-min pulse of infected cells with radioactive amino acids at 7h postinfection, gp90 was not detected, whereas gp26.5 and gp25.5 were only labeled to a small extent. During a subsequent chase period gp150 was processed to gp90, whereas the radioactivity in gp26.5 and gp25.5 increased concomitantly with a reduction of label in p24. Tunicamycin, an antibiotic which inhibits the synthesis of glycopeptides bearing N glycosidically linked oligosaccharides, prevented the appearance of gp150 in mouse hepatitis virus strain A59-infected cells. Instead, a 110,000-dalton protein accumulated. In contrast, the syntheses of the smaller viral glycoproteins gp26.5 and gp25.5 were resistant to this drug, indicating that these glycosylations were of the O glycosidical type. Although the production of infectious virus in tunicamycin-treated cells was inhibited by more than 99%, release of noninfectious viral particles continued. An analysis of these particles revealed that they lacked the peplomeric glycoproteins gp90/E2 and gp180/E2. Obviously, although the surface projections were not essential for budding of virus particles from the cells, they were required for infectivity.     

Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome

Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.     

Sulfated components of enveloped viruses.

The glycoproteins of several enveloped viruses, grown in a variety of cell types, are labeled with 35SO4(-2), whereas the nonglycosylated proteins are not. This was shown for the HN and F glycoproteins of SV5 and Sendai virus, the E1 and E2 glycoproteins of Sindbis virus, and for the major glycoprotein, gp69, as well as for a minor glycoprotein, gp52, of Rauscher leukemia virus. The minor glycoprotein of Rauscher leukemia virus is more highly sulfated, with a ratio of 35SO4- [3H]glucosamine about threefold greater than that of gp69. The G protein of vesicular stomatitis virus was labeled when virions were grown in the MDBK line of bovine kidney cells, although no significant incorporation of 35SO4(-2) into this protein was observed in virions grown in BHK21-F line of baby hamster kidney cells. In addition to the viral glycoproteins, sulfate was also incorporated into a heterogenous component with an electrophoretic mobility lower than that of any labeled with 35SO4(-2) and [3H]leucine, this component had a much greater 35S-3H ratio than any of the viral polypeptides and thus could not represent aggregated viral proteins. This material is believed to be a cell-derived mucopolysaccharide and can be removed from virions by treatment with hyaluronidase without affecting the amount of sulfate present on the glycoproteins.     

Intracellular production of virus particles and viral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect of interferon.

The effect of interferon on the biochemical properties and the maturation process of intracellular viral particles isolated from the cytoplasmic fraction of NIH/3T3 cells chronically infected with Moloney murine leukemia virus was investigated. By labeling these virions with either [35S]methionine or [3H]glucosamine, we demonstrated that they contain the same viral proteins and glycoproteins found in extracellular virions. Interferon treatment was found to reduce the rate of intracellular virus assembly. This effect was not a consequence of an interferon inhibition of viral RNA synthesis or its translation or a consequence of an interference with the posttranslational cleavage processing of viral precursor proteins, since all of these steps were not affected by interferon. However, the reduced rate of virus assembly could be attributed to the inhibition of viral protein glycosylation observed in interferon-treated cells. Nevertheless, despite this reduced rate, virus particles accumulated in interferon-treated cells. This accumulation was probably due to the strong inhibition of their final release from such cells.     

Functional Analysis of Recombinant Respiratory Syncytial Virus Deletion Mutants Lacking the Small Hydrophobic and/or Attachment Glycoprotein Gene

Respiratory syncytial virus (RSV) produces three envelope glycoproteins, the attachment glycoprotein (G), the fusion (F) protein, and the small hydrophobic (SH) protein. It had been assumed, by analogy with other paramyxoviruses, that the G and F proteins would be required for the first two steps of viral entry, attachment and fusion. However, following repeated passage in cell culture, a viable mutant RSV that lacked both the G and SH genes was isolated (R. A. Karron, D. A. Buonagurio, A. F. Georgiu, S. S. Whitehead, J. E. Adamus, M. L. Clements-Mann, D. O. Harris, V. B. Randolph, S. A. Udem, B. R. Murphy, and M. S. Sidhu, Proc. Natl. Acad. Sci. USA 94:13,961--13,966, 1997). To explore the roles of the G, F, and SH proteins in virion assembly, function, and cytopathology, we have modified the full-length RSV cDNA and used it to rescue infectious RSV lacking the G and/or SH genes. The three resulting viruses and the parental virus all contain the green fluorescent protein (GFP) gene that serves to identify infected cells. We have used purified, radiolabeled virions to examine virus production and function, in conjunction with GFP to quantify infected cells. We found that the G protein enhances virion binding to target cells but plays no role in penetration after attachment. The G protein also enhances cell-to-cell fusion, presumably via cell-to-cell binding, and enhances virion assembly or release. The presence or absence of the G protein in virions has no obvious effect on the content of F protein or host cell proteins in the virion. In growth curve experiments, the viruses lacking the G protein produced viral titers that were at least 10-fold lower than titers of viruses containing the G protein. This reduction is due in large part to the less efficient release of virions and the lower infectivity of the released virions. In the absence of the G protein, virus expressing both the F and SH proteins displayed somewhat smaller plaques, lower fusion activity, and slower viral entry than the virus expressing the F protein alone, suggesting that the SH protein has a negative effect on virus fusion in cell culture.     

Analysis of precursors to the envelope glycoproteins of avian RNA tumor viruses in chicken and quail cells.

Immune precipitation with monospecific antiserum was employed to study the intracellular synthesis of viral glycoproteins gp85 and gp37. Labeled gp85 and gp37 were detected from lysates of cells transformed with Rous sacroma virus, strain B77, after long-term labeling with radioactive glucosamine or phenylalanine. Immune precipitates prepared from lysates of cells pulse-labeled for a short time resulted in a glycoprotein of 92,000 molecular weight (gp92). This precursor was stable in B77-transformed Japanese quail cells for several hours, whereas in chicken cells it could be chased within a few hours into virion glycoproteins gp85 and gp37. Similarly, the precursor for the structural viral proteins, pr76, persisted in quail cells much longer than in chicken cells. During very short pulses or in the presence of a glucosamine block (25 mM glucosamine), the antiserum against the viral envelope glycoproteins detected a precursor of higher electrophoretic mobility of approximately 70,000 molecular weight, "p70." Fucose label entered gp92 and gp85 as well as "p70." Proteolytic treatment of virion-bound gp85 in vitro generated two discrete glycoproteins of 62,000 and 45,000 molecular weight, but did not result in an increase in the amount of gp37.     

Mosquito cells infected with vesicular stomatitis virus yield unsialylated virions of low infectivity.

Vesicular stomatitis virus propagated in and released from Aedes albopictus cells had the normal complement of viral proteins; the glycoprotein contained carbohydrate but no sialic acid. These virions had markedly reduced hemagglutinating activity and exhibited a very high ratio of physical particles to infectious virus. In vitro sialylation of vesicular stomatitis virions grown in mosquito cells resulted in a 100-fold increase in both infectivity and hemagglutination titers to levels approaching those of virus grown in BHK-21 cells. These experiments provide an example of host-controlled modification of viral infectivity.     

Six-NB-tropic murine leukemia viruses derived from a B-tropic virus of BALB/c have altered p30.

We have examined the electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels of three virion proteins of B-tropic murine leukemia virus from BALB/c and six of its NB-tropic derivatives. The gp70 protein and a 13,000-molecular-weight virion protein tentatively identified as p15 of the NB-tropic viruses migrated with the corresponding B virus proteins. However, the major internal structural protein of type C virions, p30, of all the NB-tropic viruses migrated more rapidly than the p30 of their B virus progenitor. Although this change in p30 raises the possibility that p30 may be involved in determining the N-, B-, or NB-tropism of MuLV's, it is also possible that the change accompanies but does not directly determine the change in tropsim.