p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39.

The RNA binding protein of 56 residues encoded by the extreme 3' region of the gag gene of Rauscher murine leukemia virus (MuLV) has been chemically synthesized by a solid-phase synthesis approach. Since the peptide contains a Cys26-X2-Cys29-X4-His34-X2-Cys39 sequence that is shared by all retroviral gag polyproteins which has been proposed to be a metal binding region, it was of considerable interest to examine the metal binding properties of the complete p10 protein. As postulated, p10 binds the metal ions Cd(II), Co(II), and Zn(II). The Co(II) protein shows a set of d-d absorption bands typical of a tetrahedral Co(II) complex at 695 (epsilon = 565 M-1 cm-1), 642 (epsilon = 655 M-1 cm-1), and 615 nm (epsilon = 510 M-1 cm-1) and two intense bands at 349 (epsilon = 2460 M-1 cm-1) and 314 nm (epsilon = 4240 M-1 cm-1) typical of Co(II)----(-)S- charge transfer. The ultraviolet absorption spectrum also indicates Cd(II) binding by the appearance of a Cd(II)----(-)S- charge-transfer band at 255 nm. The 113Cd NMR spectrum of 113Cd(II)-p10 reveals one signal at delta = 648 ppm. This chemical shift correlates well with that predicted for ligation of 113Cd(II) to three -S- from the three Cys residues of p10. The chemical shift of 113Cd(II)-p10 changes by only 4 ppm upon binding of d(pA)6, indicating that the chelate complex is little changed by oligonucleotide binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordination of cleavage of gag and env gene products of murine leukemia virus: implications regarding the mechanism of processing.

Mouse 3T6 cells infected with Murine Leukemia Virus (MuLV) were cloned to yield several sublines producing viruses distinct from one another with respect to the ratio of uncleaved to cleaved gag gene-coded polyprotein, Pr65gag. The virus produced by the cloned sublines also differed in the ratio of the env gene-coded protein, p15E, to its product, p12E. The two ratios, Pr65gag/p30 and p15E/p12E, were found to be highly correlated among the cloned cell lines. Velocity gradient separation of the virions produced by individual sublines, followed by polypeptide analysis, demonstrated that the particles were inhomogeneous with respect to extent of cleavage both of PR65gag and of p15E. The two cleavages were again highly correlated. These data indicate that the gag and env gene product cleavages are not independent events but are tightly coupled.

Cytoskeleton-associated Pr65gag and retrovirus assembly.

Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65gag with cytoskeletal elements in infected mouse fibroblasts. The Pr65gag associated with Nonidet P-40 (NP-40)-insoluble cytoskeletal structures appears to be subphosphorylated in comparison to NP-40-soluble Pr65gag. The association of Pr65gag with skeletal elements can be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent, or with buffers of high ionic strength. Both the skeleton-associated Pr65gag and its NP-40-soluble counterpart can be labeled with [3H]palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65gag molecules bound to skeletal elements in the infected cell appear to be more stable to proteolytic processing than NP-40-soluble Pr65gag. While the association of Pr65gag with cytoskeleton elements in the cell is neither increased nor decreased by blocking virus assembly and release with interferon, Pr65gag appears to accumulate in the cytoskeleton-enriched fraction of cells chronically infected with a temperature sensitive mutant of R-MuLV (ts 17) when such cells are grown at the nonpermissive temperature. Based on these and other results, we have proposed a model for the active role of cytoskeleton associated Pr65gag in retrovirus assembly.

Cell surface receptors for ecotropic murine viruses are different from those for xenotropic and amphotropic viruses.

The murine cell surface receptor for Rauscher murine leukemia virus was analyzed using a binding assay involving cultured murine cells and 125I-labeled envelope glycoprotein, gp70, of the virus. The binding was competitively inhibited by extracts of ecotropic murine viruses. No inhibition was observed with a xenotropic or an amphotropic murine type-C virus, a murine type-B virus, and several nonmurine retroviruses. Unlike cells producing ecotropic murine viruses, murine cell lines actively producing either an endogenous xenotropic virus or an amphotropic virus showed a significant degree of receptor activity for Rauscher murine leukemia virus gp70. When murine cells were induced with iododeoxyuridine to produce a xenotropic virus and an ecotropic virus in distinct phases, the receptor activity of the induced cells remained unchanged during xenotropic virus release, but a dramatic decrease was evident with the onset of ecotropic virus release. These findings show that ecotropic murine viruses share a common receptor on the murine cell surface and that this receptor is distinct from those for xenotropic and amphotropic murine viruses.

Binding of retrovirus-associated protein kinase and proteins to Staphylococcus aureus.

Formalin-fixed Staphylococcus aureus strain Cowan, bearing protein A, routinely used for the absorption of antigen-antibody complexes, was found to bind protein kinase activity from disrupted Moloney murine leukaemia virus (Mo-MuLV). The Wood strain of S. aureus lacking protein A also bound the kinase with similar efficiency. About 50% of the bound kinase activity, as detected by phosphorylation of casein using [gamma-32P]ATP, could be eluted from the bacterial preparation with buffer containing 0 X 5 M-KC1. Similar results were obtained with Moloney murine sarcoma virus (Mo-MuSV) strain 349 and ts110 MuSV(MuLV). The bacterial preparation was also found to bind casein kinase activity from cellular extracts of uninfected, Rauscher murine leukaemia virus (R-MuLV)-infected and Mo-MuLV-infected cells. Analysis of [3H]leucine-labelled proteins from purified virus showed selective binding to S. aureus of only two major labelled virus proteins. One virus component bound to S. aureus had the relative mobility of p15; the other polypeptide co-migrated with virus p10. Upon exposure to increased salt concentration, most of the p10 but very little of the p15 proteins were released. The S. aureus-binding proteins from ts110 Mo-MuSV and MuSV-349 revealed similar binding and elution patterns of p10 and p15 molecules. The p10 and protein kinase activity eluted from Mo-MuLV-absorbed bacteria were separated by gel filtration into a high molecular weight species, containing p10 and kinase activity, and a low molecular weight p10 monomer lacking enzymic activity.

Cleavage of Rauscher leukaemia virus (R-MuLV) Pr65gag by the Moloney leukaemia virus (M-MuLV) proteolytic activity produces the R-MuLV-specific but not the M-MuLV-specific 40 000 dalton intermediate polypeptide.

Although the Pr65gag precursor polyproteins of Moloney murine leukaemia virus (M-MuLV) and of Rauscher murine leukaemia virus (R-MuLV) have the same apparent mol. wt. by SDS--polyacrylamide gel electrophoresis (SDS--PAGE), their initial approximately 40 000 dalton intermediate cleavage products differ in mol. wt., i.e. the M-MuLV product (Pr41.5gag) is 1500 daltons larger than the R-MuLV product (Pr40gag). We took advantage of this difference to show that in vitro cleavage of R-MuLV Pr65gag by the M-MuLV proteolytic activity gives rise to R-MuLV Pr40gag and not M-MuLV Pr41.5gag. This result suggests that the specificity for cleavage of the MuLV Pr65gag is built into the substrate.

[Characterization of viral RNA in Friend tumor cells having lost their capacity to produce viral particles]. / Caractérisation de l'ARN viral dans les cellules tumorales de Friend ayant perdu leur capacité de produire des particules virales.

Cytoplasmic RNA from Friend tumoral cells which had lost their ability to produce viral particles was analyzed for its viralRNA content. A major 32S RNA corresponding to the genome of the defective SFFV virus was detected by hybridization with a synthetic DNA complementary to the Rauscher virus genome. Very low amounts of a 34S species were also found. No 38S RNA with the size of the helper virus genome was present in the cells. It was concluded that the cessation of virus production resulted from a disappearance of the helper provirus from these cells or from a block in the transcriptional process of this provirus.

Isolation of BPgp70, a fibroblast receptor for the envelope antigen of Rauscher murine leukemia virus.

A protein that avidly binds gp70, the envelope antigen of Rauscher murine leukemia virus (RMuLV), has been purified from the culture medium used for growth of BALB/c 3T3 mouse cells. Gel filtration chromatrography revealed the apparent Mr 10,000 BPgp70 was efficiently labeled when BALB/c 3T3 cells were grown in medium containing [3H]leucine, indicating a cellular origin for BPpg70. Metabolically labeled [3H]BPgp70 was not immunoprecipitated by IgG-anti RMuLV-gp&) alone, but was immunoprecipitated when gp70 was added, an indicaton of BPgp70 x gp70 complex formation. The dissociation constant estimated by immunoprecitipation agreed with the apparent Kd for binding of gp70 to BALB/c 3T3 cells. BPgp70 reversibly inhibited specific binding of 125I-labeled BMuLV-gp70 to BALB/c 3T3 cells when it was incubated with the 125I-labeled gp70 first. These data yielded a dissociation constant similar to that calculated from the immunoprecipitation data. 125I-Labeled BPgp70 also bound specifically to cells infected with RMuLV, but not to uninfected cells. Incubation of BALB/c 3T3 cells with the IgG fraction of an antiserum to BPgp70 inhibited the specific binding of 125I-labeled gp70 to these cells, but preimmune IgG did not. Complete inhibition was achieved at a less than 100:1 ratio of IgG anti-BPgp70 to gp70 binding sites.

Kinetics of synthesis and processing of precursor polypeptides of murine leukemia virus in frog oocytes following microinjection of viral RNA.

Microinjection of Rauscher murine leukemia viral RNA into living oocytes from Xenopus laevis, in contrast to cell-free systems, allowed detailed studies on the processing of newly synthesized viral precursor polypeptides. The viral messenger appeared to be stable for at least 5 days. Maximal rate of translation of 70-S virion RNA was observed 10 h after injection. The predominant translation products after a 1-h labeling period were three precursor polypeptides of Mr 77000, 75000 and 65000. Following longer labeling periods the most stable precursor polypeptide of Mr 65000 was most prominent. In addition, several intermediates of Mr 35000--60000 were observed. After about 24 h, mature viral core proteins appeared. The rate of synthesis of the 75000-Mr and 77000-Mr viral proteins decreased gradually after injection, suggesting that viral core polypeptides somehow regulated processing or synthesis of the group-specific antigen precursors. A heterogeneous group of 90000--95000-Mr polypeptides seemed to be post-translationally modified products of the 75000-Mr and 77000-Mr proteins. However, in this study no envelope-related polypeptides were synthesized, when viral RNA (70-S or 35-S) was injected into the cytoplasm or the nucleus of Xenopus oocytes.

The structural relatedness of the virus core proteins of Rauscher and Moloney murine leukaemia virus.

The virus core proteins p30, p15, pp12 and p10 of Rauscher (R-MuLV) and Moloney murine leukaemia virus (Mo-MuLV) were purified. Two-dimensional peptide maps of 3H-leucine-containing tryptic peptides as well as elution profiles from ion-exchange chromatography of tryptic peptides derived from 3H-tyrosine-labelled R-MuLV core proteins and 14C-tyrosine-labelled Mo-MuLV core proteins were compared. The results show that the p30 and p10 proteins are very similar but that p15 and pp12 exhibit significant differences.

Murine leukemia virus proteins expressed on the surface of infected cells in culture.

Infection of JLS-V9 cells in culture with Rauscher murine leukemia virus induced the appearance on the cell surface of two classes of viral proteins: Rauscher murine leukemia virus gp70, and glycoproteins related to the viral core (gag) proteins with apparent molecular weights in sodium dodecyl sulfate polyacrylamide gels of 80 x 10(3) and 95 x 10(3). The latter proteins were identified by lactoperoxidase-catalyzed iodination of the cell surface and by metabolic labeling with [(3)H]mannose followed by immunoprecipitation with an antiserum directed against the major viral core protein, p30. Tryptic peptide maps of chloramine T-iodinated proteins indicated that 80 x 10(3) - and 95 x 10(3)-molecular-weight proteins were closely related. The 95 x 10(3)-molecular-weight protein from Rauscher murine leukemia virus-infected cells had a tyrosine fingerprint which was identical to that of the 95 x 10(3)-molecular-weight gag surface polyprotein of endogenous virus-producing AKR-A cells, suggesting that expression on the cell surface of glycosylated forms of gag precursor polyproteins may not be an exclusive property of leukemic thymocytes, but a more general phenomenon in murine leukemia virus infection. Tryptic fingerprint analysis of iodinated viral and cell-bound gp70's before and after desialylation indicated a lower level of glycosylation in the cell-bound gp70 population than in virions. Analysis of only surface-iodinated gp70 showed a simple pattern of exposed tryptic peptides which was very similar in Rauscher murine leukemia virus-infected cells and in AKR-A cells.

Effect of canavanine on murine retrovirus polypeptide formation.

Canavanine is an arginine analog which is widely used to inhibit proteolytic processing of viral polyproteins. Certain results obtained with canavanine have suggested that it may have other effects. Therefore, we examined the effects of canavanine on the cell-free synthesis of murine retrovirus proteins. It was found that the electrophoretic mobility of the major gag-related cell-free product of both Rauscher murine leukemia virus (R-MuLV) and Moloney murine sarcoma virus 124 (Mo-MuSV-124) RNA was dependent on the concentration of canavanine used during translation. As the canavanine concentration was increased up to 4 mM, the apparent size of the major gag-related polypeptide also increased from 65,000 (R-MuLV RNA) or 63,000 (Mo-MuSV-124 RNA) to approximately 80,000 daltons. Additional increases in the canavanine concentration up to 12 mM did not increase the size of the gag gene product beyond 80,000 daltons. This change in electrophoretic mobility appeared to be due to a substitution of canavanine for arginine residues in the polypeptides, not to a change in their actual size. If amber suppressor tRNA and canavanine were used together during translation of Mo-MuSV-124 RNA and Mo-MuLV RNA, the results were also in agreement with this proposal. Translation experiments done with ovalbumin mRNA and mengovirus 35S RNA indicated that canavanine incorporation caused a shift in the electrophoretic mobility of ovalbumin from 43,000 to 45,000 daltons and caused the appearance of two slightly larger polypeptides in the 155,000- and 115,000- dalton regions of the mengovirus RNA cell-free product.

Effect of interferon on mouse leukaemia virus (MuLV). V. Abnormal proteins in virions of Rauscher MuLV produced in the presence of interferon.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of non-infectious particles ('interferon' virions) containing the structural proteins coded by the env and gag genes as well as additional virus polypeptides. The major glycoprotein detected in the control virions is gp71, but 'interferon' virions contain in addition an 85K mol. wt. (gp85) glucosamine-containing, fucose-deficient glycoprotein. This is recognized by antiserum to MuLV and may be related to env pr85. Surface iodination of intact virions indicates that gp71 and gp85 are the two major components of the external envelope. However, whereas in control virions gp71 associates with p15E (gp90), this complex was not detected in 'interferon' virions. Analysis of radio-labelled (3H-amino acids or iodinated) proteins from disrupted 'interferon' virions revealed the presence of 65K, 55K, 40K, 20K and 12K mol. wt. polypeptides which could be precipitated with antiserum against MuLV. There was a distinct difference in the patterns of incorporation of pulse-labelled 3H-amino acid polypeptides into virions in the presence and absence of interferon. Those polypeptides labelled in the presence of interferon and recovered in the extracellular virions in a chase with interferon appeared to have substantially fewer copies of p30 and more of gag pr55 polypeptide than the controls. These results indicate that in the presence of interferon there are changes in the proteolytic cleavage associated with virion assembly.