Synthesis and glycosylation of polyprotein precursors to the internal core proteins of Friend murine leukemia virus

Synthesis and post-translational processing of murine leukemia virus proteins were analyzed in a murine cell line (Eveline) that produces large amounts of Friend lymphatic leukemia virus. Immunoprecipitation of l-[(35)S]methionine-labeled cell extracts demonstrated that several different virus-specific proteins antigenically related to the virion core (gag) proteins p12 and p30 become radioactive within 1 min of labeling and exhibit labeling kinetics characteristic of primary translation products. The most abundant of these were proteins with molecular weights of 75,000 and 65,000. There were, in addition, two large glycosylated polyproteins with apparent molecular weights of 220,000 and 230,000, which were precipitated by antisera to p30 or p12 but not by antiserum to the major envelope glycoproteins gp69/71. Several lines of evidence, including labeling with d-[(3)H]glucosamine and binding to insolubilized lectins, suggested that the 75,000-dalton internal core polyprotein is slowly processed to form a glycoprotein with an apparent molecular weight of 93,000. On the contrary, the 65,000-dalton protein appeared to be an immediate precursor to the virion core proteins. Its processing can involve intermediates containing p30 and p12 antigens with molecular weights of 50,000 and 40,000; however, the latter did not appear to be obligatory intermediates. The detection of the 40,000-dalton protein suggested that the genes for p30 and p12 are adjacent on the viral genome. These results indicated that there are several pathways of synthesis and post-translational processing of polyprotein precursors to the gag proteins and that several of these polyproteins are glycosylated. A comparison of gag precursor processing in rapidly growing, slowly growing, and stationary cells indicated that different pathways are favored under different conditions of cell growth. Our analysis of envelope glycoprotein synthesis has confirmed the existence of two rapidly labeled 90,000-dalton glycoproteins, which appear to be precursors to the envelope glycoproteins gp69/71.     

Efficient pseudotyping of murine leukemia virus particles with chimeric human foamy virus envelope proteins.

Incorporation of human foamy virus (HFV) envelope proteins into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We report here that wild-type HFV envelope protein can pseudotype MuLV particles, albeit at low efficiency. Complete or partial removal of the HFV cytoplasmic tail resulted in an abolishment or reduction of HFV-mediated infectivity, implicating a role of the HFV envelope cytoplasmic tail in the pseudotyping of MuLV particles. Mutation of the endoplasmic reticulum retention signal present in the HFV envelope cytoplasmic tail did not result in a higher relative infectivity of pseudotyped retroviral vectors. However, a chimeric envelope protein, containing an unprocessed MuLV envelope cytoplasmic domain fused to a truncated HFV envelope protein, showed an enhanced HFV specific infectivity as a result of an increased incorporation of chimeric envelope proteins into MuLV particles.     

Influence of defective virion core proteins on RNA maturation with an avian sarcoma virus.

The RNA of the avian sarcoma virus B77 temperature-sensitive mutant LA334 was investigated using electrophoretic analysis. The RNA from mutant virus grown at the nonpermissive temperature (42degrees C) showed a heterogeneous peak between 80 and 125S, and another at about 35S. The RNA of the mutant virus grown at the permissive temperature (35 degrees C) behaved like wild-type B77 virus RNA, exhibiting a major peak at 70S. The homology between the various RNA fractions and virus-specific DNA probe was determined, indicating that mutant virus grown at the nonpermissive temperature contains relatively large amounts of nonviral-specific RNA.     

Formation of influenza virus particles lacking hemagglutinin on the viral envelope.

We investigated the intracellular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (ts) mutants (ts134 and ts61S) of influenza virus A/WSN/33. We found that at the nonpermissive temperature (39.5 degrees C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK cells were infected with these mutant viruses, they produced noninfectious virus particles at 39.5 degrees C. The efficiency of particle formation at 39.5 degrees C was essentially the same for both wild-type (wt) and ts virus-infected cells. When compared with the wt virus produced at either 33 or 39.5 degrees C or the ts virus formed at 33 degrees C, these noninfectious virus particles were lighter in density and lacked spikes on the envelope. However, they contained the full complement of genomic RNA as well as all of the structural polypeptides of influenza virus with the exception of HA. In these spikeless particles, HA could not be detected at the limit of 0.2% of the HA present in wt virions. In contrast, neuraminidase appeared to be present in a twofold excess over the amount present in ts virus formed at 33 degrees C. These observations suggest that the presence of HA is not an obligatory requirement for the assembly and budding of influenza virus particles from infected cells. The implications of these results and the possible role of other viral proteins in influenza virus morphogenesis are discussed.     

Microglial infection by a neurovirulent murine retrovirus results in defective processing of envelope protein and intracellular budding of virus particles.

The observation of murine retrovirus infection of microglial cells in brain regions expressing spongiform neurodegenerative changes suggests that these cells may play an important role in pathogenesis. To evaluate this potential in vitro, murine microglial cells were infected in mixed glial cultures with the highly neurovirulent murine retrovirus, FrCasE. The microglia were then isolated from the mixed cultures on the basis of their differential adherence and shown to be approximately 98% pure. The infected microglia expressed viral envelope protein at the plasma membrane, while viral budding was primarily intracellular. Evaluation of the viral envelope protein by immunoblotting indicated that the immunoreactive species produced was exclusively a 90-kDa precursor protein. Very little of the envelope protein was associated with particles released from these cells, and viral titers in the culture supernatant were low. Interestingly, these cells were still capable of infecting permissive target cells when seeded as infectious centers. This partially defective infection of microglial cells suggests a potential cellular means by which a neurovirulent retrovirus could disrupt normal microglia and in turn central nervous system motor system functioning.     

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.     

Structure of the murine leukemia virus envelope glycoprotein precursor.

The glycosylated env gene precurosr (Pr80env) of Moloney murine leukemia virus has been isolated by selective immunoprecipitation. Use of the drug tunicamycin to inhibit nascent glycosylation or specific cleavage with endoglycosidase H demonstrated that the precursor contained an apoprotein with a molecular weight of 60,000. The finished virion glycoprotein (gp70) was largely resistant to the action of endoglycosidase H. Chromatography of the glycopeptides of Pr80env in conjunction with endoglycosidase H digestion studies suggested that the precursor contained two distinct major glycosylation sites. Analysis of partial proteolytic cleavage fragments of Pr80env before and after endoglycosidase H treatment placed the two glycosylation sites within a 30,000-dalton region of the apoprotein sequence. Kinetic experiments showed that carbohydrate processing as well as proteolytic cleavage are late steps in the maturation of Pr80env.     

In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).     

Structure of the intracellular defective viral RNAs of defective interfering particles of mouse hepatitis virus.

The intracellular defective RNAs generated during high-multiplicity serial passages of mouse hepatitis virus JHM strain on DBT cells were examined. Seven novel species of single-stranded polyadenylic acid-containing defective RNAs were identified from passages 3 through 22. The largest of these RNAs, DIssA (molecular weight [mw], 5.2 X 10(6)), is identical to the genomic RNA packaged in the defective interfering particles produced from these cells. Other RNA species, DIssB1 (mw, 1.9 X 10(6) to 1.6 X 10(6)), DIssB2 (mw, 1.6 X 10(6)), DIssC (mw, 2.8 X 10(6)) DIssD (mw, 0.82 X 10(6)), DIssE (mw, 0.78 X 10(6)), and DIssF (mw, 1.3 X 10(6)) were detected at different passage levels. RNase T1-resistant oligonucleotide fingerprinting demonstrated that all these RNAs were related and had multiple deletions of the genomic sequences. They contained different subsets of the genomic sequences from those of the standard intracellular mRNAs of nondefective mouse hepatitis virus JHM strain. Thus these novel intracellular viral RNAs were identified as defective interfering RNAs of mouse hepatitis virus JHM strain. The synthesis of six of the seven normal mRNA species specific to mouse hepatitis virus JHM strain was completely inhibited when cells were infected with viruses of late-passage levels. However, the synthesis of RNA7 and its product, viral nucleoprotein, was not significantly altered in late passages. The possible mechanism for the generation of defective interfering RNAs was discussed.     

Biochemical and mutational analysis of a plant virus polyprotein cleavage site.

The RNA genome of tobacco etch virus (TEV) is organized as a single translational unit coding for a 346,000 (346 kd) mol. wt (Mr) polyprotein. The 346 kd Mr polyprotein is cleaved by a 49 kd Mr virus-encoded proteinase at five different sites between the dipeptides Gln-Ser or Gln-Gly. These cleavage sites or gene product boundaries are defined by the heptapeptide sequence...Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly.... We have used the 54 kd Mr nuclear inclusion protein/30 kd Mr capsid protein junction as a model to examine the role of these conserved amino acids in defining a cleavage site. The 54 kd/30 kd Mr protein cleavage site sequence of 10 TEV isolates from geographically distinct locations has been deduced. The conserved amino acids are present in all isolates. To determine if these four amino acids are an absolute requirement for polyprotein substrate activity, a site-directed mutational analysis has been performed. A recombinant cDNA molecule encoding the TEV 54 kd/30 kd Mr gene product cleavage site was mutated and polyprotein substrates were synthesized and processed in a cell-free system. Single amino acid substitutions made at the different positions reveal a strong preference for the naturally conserved amino acids.     

Clathrin-mediated endocytosis and lysosomal cleavage of hepatitis B virus capsid-like core particles

The hepatitis B virus (HBV) core particle serves as a protective capsid shell for the viral genome and is highly immunogenic. Recombinant capsid-like core particles are used as effective carriers of foreign T and B cell epitopes and as delivery vehicles for oligonucleotides. The core monomer contains an arginine-rich C terminus that directs core particle attachment to cells via membrane heparan sulfate proteoglycans. Here we investigated the mechanism of recombinant core particle uptake and its intracellular fate following heparan sulfate binding. We found that the core particles are internalized in an energy-dependent manner. Core particle uptake is inhibited by chlorpromazine and by cytosol acidification known to block clathrin-mediated endocytosis but not by nystatin, which blocks lipid raft endocytosis. Particle uptake is abolished by expression of dominant negative forms of eps15 and Rab5, adaptors involved in clathrin-mediated endocytosis and early endosome transport, respectively. Endocytosed particles are transported to lysosomes where the core monomer is endoproteolytically cleaved into its distinct domains. Using protease inhibitors, cathepsin B was identified as the enzyme responsible for core monomer cleavage. Finally we found that monomer cleavage promotes particle dissociation within cells. Together, our results show that HBV capsid-like core particles are internalized through clathrin-mediated endocytosis, leading to lysosomal cleavage of the core monomer and particle dissociation.     

Identification of proteins encoded by the Gazdar murine sarcoma virus genome by in vitro translation and comparison with Moloney murine sarcoma virus 124.

The gene products of Gazdar murine sarcoma virus (Gz-MuSV) were identified by in vitro translation of Gz-MuSV virion RNA. An overlapping set of proteins with approximate molecular weights of 37,000 (37K), 33K, 24K, and 18K were synthesized from the transforming gene of Gz-MuSV, v-mosGz. In addition, Gz-MuSV-specific RNA directed the in vitro synthesis of a 62K gag gene protein and a 37.5K env gene-related product. The Gz-MuSV-specific in vitro translation products were compared with the in vitro translation products of M-MuSV 124, an independent isolate with a similar v-mos gene. This analysis showed that the 62K Gz-MuSV gag gene protein and the 37K, 33K, 24K, and 18K v-mosGz proteins were almost identical to the M-MuSV 124 62K (gag) and 37K, 33K, 24K, and 18K (v-mosMo) proteins that we previously identified and characterized. The 37.5K env gene product from Gz-MuSV does not have a correlate in the M-MuSV 124 translation products. These results were analyzed in the context of expectations based on similarities and differences in genetic organization of these two viral genomes.     

Incorporation of homologous and heterologous proteins into the envelope of Moloney murine leukemia virus.

The efficiencies with which homologous and heterologous proteins are incorporated into the envelope of Moloney murine leukemia virus (M-MuLV) have been analyzed by utilizing a heterologous, Semliki Forest virus-driven M-MuLV assembly system and quantitative pulse-chase assays. Homologous M-MuLV spike protein was found to be efficiently incorporated into extracellular virus particles when expressed at a relatively low density at the plasma membrane. In contrast, efficient incorporation of heterologous proteins (the spike complex of Semliki Forest virus and a cytoplasmically truncated mutant of the human transferrin receptor) was observed only when these proteins were expressed at high densities at the cell surface. These results imply that homologous and heterologous proteins are incorporated into the M-MuLV envelope via two distinct pathways.     

Complementation studies with Rous sarcoma virus gag and gag-pol polyprotein mutants.

Avian retroviruses (with the notable exception of spleen necrosis virus) express their protease (PR) both in their gag and their gag-pol polyprotein precursors, in contrast to other retroviruses, notably, the mammalian retroviruses, in which PR is encoded in the gag-pol polyprotein or in a separate reading frame as a gag-pro product. The consequence is that the avian PR is expressed in stoichiometric rather than catalytic amounts. To investigate the significance of the particular genome organization of the avian retrovirus prototype Rous sarcoma virus, we developed an assay that measures complementation between the gag and the gag-pol polyproteins by expressing them from two different plasmids in transfected cells. By using this assay, we showed that the protease PR from the gag-pol polyprotein is capable of autocatalytic self-cleavage and -activation when coexpressed with a protease-deficient gag protein and that the PR domain has a role in viral particle assembly. Furthermore, this complementation assay can be used to investigate the role of the gag domain in the gag-pol polyprotein by determining whether it can rescue a defect in the gag polyprotein. We report here the results of such an experiment, which studied a mutation in the N terminus of the gag gene.     

Functional characterization of the N termini of murine leukemia virus envelope proteins

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.     

Topography of murine leukemia virus envelope proteins: characterization of transmembrane components

Trypsinization of intact Moloney murine leukemia virus resulted in cleavage of p15(E) and Pr15(E) at a site near the middle of the molecule, producing a 9,000-dalton amino-terminal fragment which contains the disulfide linkage site to gp70 and which carries p15(E) epitopes b and c, but not epitope a. After solubilization of the viral membrane, trypsinization occurred at a second site within 1,000 daltons of the carboxy end of p15(E). This site is not exposed in intact virions, indicating that p15(E) and Pr15(E) are transmembrane proteins.     

Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein.

We describe the construction of a plasmid (pCAT2AGUS) encoding a polyprotein in which a 19 amino acid sequence spanning the 2A region of the foot-and-mouth disease virus (FMDV) polyprotein was inserted between the reporter genes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS) maintaining a single, long open reading frame. Analysis of translation reactions programmed by this construct showed that the inserted FMDV sequence functioned in a manner similar to that observed in FMDV polyprotein processing: the CAT2AGUS polyprotein underwent a cotranslational, apparently autoproteolytic, cleavage yielding CAT-2A and GUS. Analysis of translation products derived from a series of constructs in which sequences were progressively deleted from the N-terminal region of the FMDV 2A insertion showed that cleavage required a minimum of 13 residues. The FMDV 2A sequence therefore provides the opportunity to engineer either whole proteins or domains such that they are cleaved apart cotranslationally with high efficiency.