An avian oncovirus mutant deficient in genomic RNA: characterization of the packaged RNA as cellular messenger RNA.

SE 21Qlb is a recombinant avian oncovirus which is continuously produced by a line of transformed quail cells. These cells produce no detectable infectious viral progeny. Virions of SE 21Q1b contain 1–2% of the normal amounts of RSV-specific RNA found in PR-RSV-C virions and substantial quantities of heterogeneously sedimenting nonviral RNA (M. Linial, E. Medeiros, and W. S. Hayward, 1978b, Cell, 15:1371–1381). RNA extracted from purified virions of SE 21Q1b is as efficient a template for amino acid incorporation in a mRNA-dependent reticulocyte lysate as oligo(dT)-cellulose purified cellular mRNA. Virion RNA from SE 21Q1b serves as a template in vitro for a small amount of the precursor to the internal structural proteins (gag proteins). But unlike RNA from other avian oncoviruses, SE 21Q1b RNA makes, in addition, a number of proteins whose sizes on sodium dodecyl sulfate (SDS)-polyacrylamide gels resemble those of a number of uninfected quail fibroblast proteins. The proteins range in size from 15,000–220,000 daltons. Immunoprecipitation of proteins synthesized in vitro from SE 21Qlb virion RNA with antisera against the gag, pol, and env gene products shows that this RNA serves as a template for only one viral gene product, the recombinant gag gene precursor of 72,000 daltons (ΔPr76) (R. Shaikh, M. Linial, J. Coffin, and R. Eisenman, 1978, Virology87, 326–338). Addition of lysed virus to proteins made in vitro from PR-C RNA or RNA from PR-E-95c, a recombinant whose gag gene structure is similar to that of PR-E-21Qlb, results in the cleavage of gag protein precursors (K. von der Helm, 1977, Proc. Nat. Acad. Sci. USA74, 911–915) into several of the internal structural proteins. On the other hand, products of translation of PR-E-21Q1b RNA are not detectably cleaved into the internal structural proteins, suggesting that a very low amount of PR-E-21Q1b translation products are gag related. A protein of 37,000 daltons constitutes about 15% of the total protein synthesized from SE 21Qlb RNA. Data from serological and peptide mapping studies indicate that this protein is unrelated to the virion gag, pol, or env proteins. However, the major tryptic peptide of this protein appears to be identical to the major peptide of a prominent quail cell protein having the same apparent molecular weight as the in vitro translation product. Thus, several lines of evidence suggest that SE 21Q1b virions contain substantial amounts of functional cellular messenger RNAs.     

Recombinant avian oncoviruses. I. Alterations in the precursor to the internal structural proteins.

The synthesis of the gag precursor protein (Pr76) was studied in a number of recombinant avian oncoviruses, which were selected for recombination between the env and pol genes or the env and src genes. Such studies show that the electrophoretic mobility of the gag precursor protein of recombinant viruses (ΔPr76) was greater than that of the parental gene product (Pr76) in 16 of 24 cases. Viruses derived from recombination between endogenous (RAV-0) and exogenous viruses (RSV), as well as between two exogenous viruses, showed the ΔPr76 phenotype. In an mRNA-dependent rabbit reticulocyte translation system, 35 S RNA isolated from PR-RSV-C directed the synthesis of Pr76, while RNA isolated from a recombinant between PR-RSV-C and RAV-0 directed the synthesis of ΔPr76. These observations show that the synthesis of ΔPr76 is due to an alteration in the genome related to recombination. An analysis of the RNase T₁-resistant oligonucleotides demonstrated a crossover near the 5′ end of the genome (which may be within the gag gene) in two recombinant virus clones which synthesize ΔPr76 in infected cells; but no crossover was detected near the 5′ end of the genome in a third recombinant virus clone which synthesizes Pr76 in infected cells. Our data suggest that the synthesis of ΔPr76 is a consequence of recombination near the 5′ end of the genome.     

Continuous production of Rous sarcoma virus free of transformation defective virus in clones of established RSV-transformed quail cells

Quail embryo fibroblasts were infected with a Schmidt-Ruppin strain RSV × chf recombinant virus. Virus-transformed cells were established as a permanent line and then cloned in methyl cellulose. Out of 140 clones isolated four clones were capable of indefinite growth. These clones were examined for (i) production of sarcoma and td virus particles, (ii) number of integrated virus genome equivalents, and (iii) deletions of the src gene in the provirus. We found that the clones yield about 106 focus-forming units of the sarcoma virus per milliliter of the culture medium. No td virus could be detected by plating of the virus at the endpoint dilution and no 35 S td virus RNA but only 38 S sarcoma virus RNA was found in virions. Hybridization kinetic studies indicated that three different clones contain about 2 virus genome equivalents, and one clone contains about 4 virus genome equivalents per diploid cell. Upon transfection the proviruses of different clones generated sarcoma viruses and no td viruses. Finally digestion with EcoRI restriction endonuclease released in all four clones a 1.9 × 106-dalton fragment characteristic of the complete src gene, while no 0.8 × 10⁶-dalton fragment characteristic of a td provirus could be detected. We concluded that the clones of RSV-transformed quail cells contain only nondefective sarcoma proviruses and produce from these proviruses nondefective focus-forming virions in the absence of any segregant td virions.     

A comparison of "influenza C" with prototype myxoviruses: receptor-destroycing activity (neuraminidase) and structural polypeptides.

“Influenza C” virus agglutinates chicken erythrocytes and elutes rapidly from them, in the process destroying receptors for the virus. Virions of influenza C purified by centrifugation into a density gradient retain the ability to agglutinate and elute from red blood cells and to destroy their receptors for influenza C. However, the receptor-destroying activity of influenza C does not affect receptors for prototype ortho- and paramyxoviruses. Purified and concentrated bacterial neuraminidase destroys receptors for influenza A and B and Newcastle disease viruses without affecting receptors for influenza C. These results confirm and extend previous observations by Hirst that influenza C receptors differ from receptors for myxoviruses and suggest they do not contain sialic acid. Direct assay for neuraminidase activity in “influenza C” was carried out employing as substrates compounds containing predominantly N-acetyl, 4-O-acetyl N-acetyl, or 7-O-acetyl and 8-O-acetyl N-acetyl neuraminic acid in 2–3′-, 2–4′-, 2–6′- and 2–8′-α-O-glycosidic linkage. Although differences in specificity were observed between Vibrio cholerae neuraminidase, influenza A and B neuraminidase, and Newcastle disease virus neuraminidase, each neuraminidase was active against several of the substrates whereas “influenza C” did not liberate sialic acid from any substrate. It is concluded that “influenza C” lacks an α-neuraminidase. By acrylamide-gel electrophoretic analysis in the presence of sodium dodecyl sulfate influenza C virions were found to contain three glycoproteins, of sizes about 83,000, 66,000 and 26,000 daltons. The main structural polypeptide was nonglycosylated, had a size of about 28,000 daltons, and was the only polypeptide present in enveloped structures recovered after prolonged proteolytic digestion which destroys all morphologically identifiable components of influenza C virions except for the structural envelope. This component is thus identified as the internal membrane protein. An additional non-glycosylated major polypeptide component in virions, of size about 62,000 daltons, is believed to be the nucleoprotein. The pattern of structural polypeptides is distinct from that of paramyxoviruses, but in some respects similar to orthomyxoviruses. Classification and nomenclature of “influenza C” virus are discussed.     

Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity of proteolytic cleavage of an inactive precursor protein of Sendai virus

The glycoproteins of Sendai virus have been isolated by a procedure involving extraction with the nonionic detergent Triton X-100 and affinity chromatography on fetuin-Sepharose. The largest Sendai virus glycoprotein (MW ~69,000) possesses both hemagglutinating and neuraminidase activities, and has been designated HN.Virions grown in MDBK cells or in the allantoic sac of the chick embryo contain similar amounts of the HN glycoprotein, but differ in their content of the other glycoproteins. Virions grown in MDBK cells contain a large amount of a glycoprotein designated F₀ (MW ~65,000). This glycoprotein is a precursor of a smaller virion glycoprotein, F (MW ~53,000) which is present in only small amount in MDBK cellgrown virions. F₀ can be cleaved to yield F by treatment of virions with trypsin in vitro. Sendai virions grown in the chick embryo lack the precursor protein F₀, but contain a large amount of F; in this case, proteolytic cleavage of F₀ to yield F occurs in ovo.The Sendai virions grown in MDBK cells, which are deficient in the small glycoprotein F, lack hemolyzing and cell-fusing activities and cannot infect MDBK cells. Virions grown in the chick embryo, which contain much F, possess both these activities and can infect MDBK cells as well as the chick embryo. MDBK cell-grown virions acquire hemolyzing and cell-fusing activities and become infective for MDBK cells when the precursor glycoprotein F₀ is cleaved in vitro to yield F.The results indicate that the small glycoprotein of paramyxoviruses is biologically active and is involved in virus-induced hemolysis, cell fusion, and the initiation of infection. The precise mechanism by which this glycoprotein participates in these reactions remains to be determined, but is now amenable to experimentation. The precursor of this glycoprotein is biologically inactive, but is incorporated into virions grown in some host cells; it may be activated by proteolytic cleavage either in vivo or in vitro. The present results provide a biochemical basis for previously observed host-dependent variation in infectivity, and in hemolysis and cell-fusion induced by paramyxoviruses.     

Low freqeuncy production of recombinant subgroup E avian leukosis viruses by uninfected V-15B chicken cells.

15_B chicken contains V-15_B an allele for the spontaneous production of a noninfectious avian leukosis virus, 15_B-ILV. In this paper we present evidence that uninfected 15_B cells also produce subgroup E viruses, designated 15_B-E viruses. In contrast to 15_B-ILV which occurs at an estimated frequency of one event per 10³ 15_B cell days, 15_B-E viruses occur at estimated frequencies of one event per 2 × 10⁹ cell days. The frequency of occurrence of 15_B-E viruses was affected by the level of expression of V-15_B and the presence of endogenous viral alleles which constitutively express subgroup E envelope antigens. The proteins of nine independent isolates of 15_B-E viruses were analyzed by SDS-polyacrylamide gel electrophoresis. All isolates had p27 proteins with the distinctive electrophoretic mobility of the p27 of RAV-0, an endogenous virus. Each isolate had characteristic pairs of p19 related proteins which were similar to pairs of p19 related proteins, which are generated by recombination of PR-RSV-C and RAV-0. Since 15_B-E isolates occurred in uninfected cells and contained proteins which are characteristic of RAV-0 and recombinants of RAV-0, we suggest that 15_B-E viruses are recombinants of endogenous virus information. Since the level of expression of V-15_B and the presence of endogenous viral alleles which constitutively express subgroup E envelope antigens affected the frequency of occurrence of 15_B-E viruses, we further suggest that 15_B-ILV and RNAs which contain information for subgroup E envelope antigens are the parents of 158E viruses.     

Proteins specified by group B togaviruses in mammalian cells during productive infections

Seven nonstructural and two of the three virion proteins specified by each of Kunjin, dengue type 2, St. Louis encephalitis, and Japanese encephalitis virus were labeled during replication in Vero and in PS cells which had been treated only with actinomycin. After host protein components were eliminated from electrophoretic profiles of infected cytoplasm using a double-label and subtraction method, all virusspecified proteins were readily identified. All profiles are similar but distinguishable from one another. The proteins are synthesized in the same but not in equimolar proportions for long periods. Molecular weight of the major core protein is 13,500 daltons, but the envelope protein ranges from 51,300 daltons (Kunjin) to 59,000 daltons (dengue). The major core protein of the Kunjin virion appeared to be either unstable in cytoplasm or was rapidly lost by incorporation into virions. No evidence was obtained for posttranslational cleavage; the information encoded in the RNA genome of 4.2 × 10⁶ daltons is sufficient to account for the total of about 370,000 daltons of virus-specified proteins.     

Electrophoretic analysis of the RNA of avian tumor viruses

The heat-dissociated RNA of avian leukosis and sarcoma viruses of several subgroups was analyzed by polyacrylamide gel electrophoresis. Heterogeneous patterns obtained in early experiments appeared to be due primarily to RNA degradation during steps of RNA characterization by sucrose gradient centrifugation rather than procedures of virus purification, virus incubation at 37°, or RNA extraction. All the viruses studied contained a major RNA species with an estimated size between 2.5 and 2.8 × 10⁶ daltons and at least one minor RNA species of slightly smaller size. This suggests that if the genome of avian tumor viruses is indeed segmented, the subunits are of a similar size. The heat-dissociated RNA of RAV-2, RAV-60, RAV-50, and Schmidt-Ruppin RSV (subgroup A) was generally less complex than that of Schmidt-Ruppin RSV (subgroup D). No significant difference was found in the RNA of RAV-2 grown in chicken cells that were positive or negative for the expression of latent viral genes (chf). However, in the absence of other known leukosis viruses, the pattern of heat-dissociated RNA of Bryan RSV grown in cells expressing chf functions was consistently broader than that of Bryan RSV grown in cells not expressing chf functions.     

Identification and characterization of a structural protein of hepatitis B virus: a polymerase and surface fusion protein encoded by a spliced RNA

The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using (32)P-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.     

Mutants of Rous sarcoma virus with extensive deletions of the viral genome.

Deletion mutants of Rous sarcoma virus (RSV) have been isolated from a stock of Prague RSV which had been irradiated with ultraviolet light. Quail fibroblasts were infected with irradiated virus and transformed clones isolated by agar suspension culture. Three clones were obtained which did not release any virus particles. Analysis of DNA from these non-producer clones with restriction endonucleases and the Southern DNA transfer technique indicated that the clones carry defective proviruses with deletions of approximately 4 × 10⁶ daltons of proviral DNA. The defective proviruses, which retain the viral transformation (src) gene, contain only 1.7–2.0 × 10⁶ daltons of DNA. Multiple species of viral RNA containing the sequences of the src gene were detected in these clones; some of these RNAs may contain both viral and cellular sequences. The protein product of the src gene, p60^src (Brugge and Erikson, 1977), was also synthesized in the nonproducer clones. However these clones did not contain the products of the group-specific antigen (gag), DNA polymerase (pol), or envelope glycoprotein (env) genes, nor did they contain the 35 and 28 S RNA species which are believed to represent the messengers for these viral gene-products. The properties of these mutants indicate that expression of the src gene is sufficient to induce transformation. These clones may represent useful tools for the study of the expression of this region of the genome.     

Characterization of two conditional early mutants of Rous sarcoma virus

Some of the biological and physical properties of two independently isolated mutants of Rous sarcoma virus have been investigated. Both ts 335 and ts 337 are unable either to transform or replicate at 41 °. The lesions in the mutants occur very early after infection (prior to 2 hr); however, the lesions appear to be expressed after adsorption to and penetration into the host cells. Both mutants contain temperature sensitive structural components, and ts 337 is also antigenically different from the wild type. The virions contain a polymerase activity which appears to be temperature sensitive in both the endogenous reaction and with poly rA:d(pT)₁₀ as the exogenous template. Upon coinfection of chick fibroblasts with ts 335 or ts 337 and Rous-associated virus (RAV), genetically stable wild-type sarcoma virus is produced at a high frequency. Therefore, the information for the function(s) which is mutant in these viruses is also present in RAV. All the parameters measured are consistent with a mutation affecting the function of the avian tumor virus RNA-dependent DNA polymerase.     

Infection of chick cells by subgroup E viruses.

Studies were carried out to determine whether there is a restriction of replication of the endogenous chicken leukosis virus Rous associated virus type O (RAV-0) in chick embryo fibroblasts (CEF) beyond that imposed by the known cell surface barrier. Following either Sendai virus mediated fusion of RAV-O with surface resistant CEF ($\text{C}\text{E}$ cells), or infection of CEF lacking the surface barrier ($\text{C}\text{O}$ cells), a quantitative 10³- to 10⁴-fold restriction in replication was noted in comparison with RAV-60, a recombinant leukosis virus bearing the same subgroup E envelope as RAV-0 The failure of this internal restriction to operate against subgroup E leukosis viruses was investigated in detail in a series of cloned subgroup E sarcoma virus recombinants isolated following mixed infection with RAV-0 and Prague strain of Rous sarcoma virus subgroup C (PR-C) (i.e., two factor crosses) or following PR-C infection of chf⁺ CEF. RNA from one of these PR-E clones was shown to contain a nearly full complement of RAV-0 specific and RSV specific nucleotide sequences, but that isolate and six others were all free of the restriction observed with RAV-0 replication. One clone may be subject to some restriction. Thus some genetic function(s) of RAV-0 lost or inoperative in recombinant viruses appears important for this restriction. Since RAV-0 can replicate to a “normal” titer in some CEF from particular lines of chickens (line 7, line 100 × 7, and line 15), but apparently not in embryo culture from chicken flocks used in these studies, some cell function(s) also appears important for this restriction.     

Altered virion proteins of a temperature-sensitive mutant of polyoma virus, ts59.

The ts59 mutant of polyoma virus is blocked in a late step of infection at the restrictive temperature. Cellular and viral DNA synthesis proceed normally in ts59-infected cells at the restrictive temperature, but infectious progency virus particles are not assembled. The ts59 mutant complements early tsA mutants in mixed infection, and the temperature-sensitive mutation maps in the late region of the polyoma genome. The infectivity of ts59 virions is much heat labile than wild-type polyoma. All three nonhistone capsid proteins of ts59, VP1 (45,000 daltons) and the overlapping proteins VP2 (30,000 daltons) and VP3 (20,000 daltons), show altered mobilities when analyzed by SDS-polyacrylamide gel electrophoresis. The tryptic peptide patterns of all three ts59 virion proteins also differ from the tryptic peptide patterns of wild-type proteins. Analysis of the ts59 proteins synthesized in vitro and in infected cells suggests that the alterations in the ts59 virion proteins are caused by differences in primary structure rather than by post-translational modifications. The capsid proteins of convertant virions produced by marker rescue of the ts59 temperature-sensitive mutation, using various restriction endonuclease fragments of wild-type DNA, have been analyzed. Results of these studies suggest that (i) 26 map units is the furthest point, in a clockwise direction on the genetic map, that the information for the C-terminus of VP1 can be from the Eco·R1 cleavage site; (ii) the N-terminal end of VP2 extends beyond the N-terminal end of VP3; (iii) the temperature-sensitive phenotype of ts59 is correlated with a peptide alteration common to VP2 and VP3. The ts59 mutant contains two further peptide alterations not related to the temperature-sensitive phenotype: a C-terminal alteration in VP1 and an alteration unique to VP2. Cells infected by ts59 contain approximately fourfold lower amounts of viral capsid proteins and virus-specific messenger RNA at the restrictive temperature compared to the permissive temperature.     

Recombinant avian oncoviruses. II. Alterations in the gag proteins and evidence for intragenic recombination.

The internal structural (gag) proteins of recombinant avian oncoviruses selected for the env gene of RAV-O (an endogenous chicken virus) and the src gene for PR-RSV-C were examined. Eight of ten clones of such recombinants were found to synthesize altered gag proteins. The gag proteins of one recombinant clone, PR-E-95c, were examined in more detail by gel electrophoresis and tryptic peptide mapping. These methods have allowed us to distinguish between the gag proteins of the two parental viruses and to determine from which virus the proteins of the recombinant virus were derived. PR-E-95c virions were found to contain p27₀, an electrophoretically distinguishable variant of p27 which is found in isolates of RAV-0. This recombinant virus also contains p12/15, which is electrophoretically indistinguishable from the p12/15 of both of the parental viruses. However, tryptic peptide analysis of p15 indicates that PR-E-95c has inherited PR-RSV-C-specific p15 sequences. These observations suggest that at least one cross-over has occurred between p15 and p27 in PR-E-95c. A striking difference between the proteins of PR-E-95c virus and those of the parental viruses is that the recombinant lacks polypeptides migrating in the position of p19 and contains two novel polypeptides termed p19α (MW 20,000) and p19β (MW 15,000). Both of these polypeptides are phosphorylated and share antigenic determinants and some tryptic peptides with parental p19. As determined by peptide analysis and radioimmunoassay, these p19-related proteins contain information from both parental viruses, suggesting that PR-E-95c has another cross-over within p19. The altered p19 proteins bind to viral RNA specifically and are associated with genomic RNA in the virion. Neither the stability nor the specific infectivity of the recombinant viruses appears to be significantly affected by the altered proteins.     

Phosphoproteins of Rous sarcoma viruses.

³²P-labeled proteins from Rous sarcoma viruses were extracted with phenol and analyzed by SDS-polyacrylamide gel electrophoresis. p19 was found to be the major phosphorylated protein. p12, a ribonucleoprotein, was also phosphorylated. Phosphorylation took place at both serine and threonine. The phosphorylation patterns of viral proteins were studied with respect to subgroup specificity, transforming ability, and maturation ability of the viruses. It was found that some of the viruses belonging to subgroup A, including RAV-3, RSV(RAV-1), and PR-B:RAV-3, contain a novel p23 phosphoprotein in addition to phosphorylated p19 and p12. It could not be determined whether p23 phosphoprotein was cellular or viral in origin. The phosphorylation patterns did not vary with regard to other viral properties. Furthermore, the in vivo phosphorylated proteins were found to be different from the viral proteins phosphorylated in vitro by virion-associated protein kinases. It was suggested that the phosphorylation of viral proteins in vivo is a specific process.     

Replication of influenza virus at elevated temperatures: Production of virus-like particles with reduced matrix protein content

The average matrix protein content in virions of wild-type influenza A/Ann Arbor/6/ 60 (H2N2) or A/Queensland/6/72(H3N2) is reduced by about half when virus is grown in chick kidney cells at 39° instead of at 34°. This results from the production at 39° of a subpopulation of virions having a low buoyant density (P = 1.14 g/cm³) and having as matrix protein only about 5 to 10% of their total protein, in contrast to normal virions with a matrix protein content of approximately 50% and a buoyant density of 1.20 g/cm³. Although the virions comprising the low density subpopulation have little matrix protein, they contain approximately normal proportions of hemagglutinin and nucleoprotein. This suggests that matrix protein may not be essential for maturation of influenza virions containing nucleoprotein and viral modified membrane. However, as judged by their morphology, the virions with a reduced matrix protein content appeared more fragile than normal virions, which implies that a major role for matrix protein is maintaining the structural integrity of influenza virus particles.     

RNA specific for the transforming component of avian erythroblastosis virus strain R.

Avian erythroblastosis virus strain R (AEV) contains two species of RNA, 35 S and 30 S, when rescued from nonproducing AEV-transformed chicken cells as pseudotypes of Rous-associated virus-1 (RAV-1) or of AEV-associated virus (REAV). Biological and electrophoretic data suggest that the 30 S RNA is specific for the transforming component of AEV. In fingerprint analysis of RNase T₁-resistant oligonucleotides the 30 S RNA of AEV(RAV-1) yields 13 distinct oligonucleotide spots, 7 of which are unique to the 30 S RNA of AEV, the others are common to RAV-1. The fingerprint of 30 S RNA obtained from AEV(REAV) revealed a similar pattern of unique and shared oligonucleotides. These data suggest that the genome of AEV strain R contains specific genetic sequences which may code for a product that causes transformation of the host cell.