The origin of 7 S RNA in virions of avian sarcoma viruses.

Duck and chick embryo fibroblasts were labeled with ³²PO₄^3? and cellular 7S RNA was purified. Ribonuclease T₁ digests were prepared and the resulting oligonucleotides were fractionated by two-dimensional electrophoresis. Duck 7 S RNA lacked one large oligonucleotide present in chicken 7 S RNA, thus establishing that the two species contain 7 S RNAs of different sequence. The Prague C strain of Rous sarcoma virus was grown in each type of host and the 7 S RNA found in progeny virus was examined. It was found to be identical to that of the host which produced the virus.     

The polypeptides of canine distemper virus: Synthesis in infected cells and relatedness to the polypeptides of other morbilliviruses

The biochemical and immunological properties of the polypeptides of canine distemper virus (CDV), their synthesis and processing in infected cells, and their relatedness to the polypeptides of other morbilliviruses have been studied. CDV virions contain six major polypeptides which are analogous to those of measles virus (MV). These polypeptides with their estimated molecular weights (m_r) are: L (200,000); H (76,000); P (66,000); NP (58,000); F (62,000), which consists of two disulfide-linked polypeptides, F₁ (40,000) and F₂ (23,000); and M (34,000). The H, F₁, and F₂ polypeptides of CDV are glycosylated; the presence of carbohydrate on F₁ is in contrast to its absence on the F₁ of MV. The CDV F₂ has a larger apparent M_r than the MV F₂ (23,000 vs 12,000). The NP and P polypeptides of CDV are phosphorylated, and in pulse-chase experiments in CDV-labe;ed cells the P polypeptide was rapidly lost. In addition to the structural polypeptides, a putative nonstructural protein, NS (M_r 18,000), was found in CDV-infected cells. The polypeptide also turned over rapidly in pulse-chase experiments.The immunological relatedness of the polypeptides of MV and CDV and of two other morbilliviruses, rinderpest (RV) and a bovine encephalitis virus (107) was shown by immuno-precipitation of the viral polypeptides from CDV- and MV-infected cells with antisera against each of the four viruses. The only failure to exhibit reciprocal reactivity between CDV and MV was found with the H polypeptides, where only a one-way cross was found, i.e., MV antiserum precipitated all of the CDV polypeptides, whereas CDV antiserum precipitated all of the MV polypeptides except H. RV antiserum resembled that of MV; it precipitated all of the polypeptides of both MV and CDV, whereas 107 antiserum, like that of CDV, precipitated all of the CDV polypeptides and all of the MV polypeptides except H. These results indicate that these four morbilliviruses with different host ranges are antigenically closely related, with MV apparently more closely related to RV, and CDV to 107 virus. In spite of their antigenic similarities, the individual polypeptides of CDV and MV could be easily distinguished by peptide mapping. Some similarities were found in the internal polypeptides P, NP, and M, but very little in the surface glycoproteins, H and F₁.     

Genetic diversity among avian influenza viruses

The RNAs of a series of avian influenza viruses of the subtype Hav7Neg2 were examined to determine if their antigenic similarity reflected an overall conservation of their RNA sequences. Genetic analysis by gel electrophoresis showed a marked variability in all of the RNA segments of the isolates. Analysis by competitive hybridization indicated that with some genome segments this variability represented major genetic differences. These differences were present even among concurrent isolates from one geographical area. In contrast, similar analysis of human H3N2 influenza virus isolates showed that viruses isolated 9 years apart were much more similar than the cocirculating avian viruses. The genetic diversity in avian influenza viruses may result from the cocirculation of many different influenza A viruses in ducks and their ability to recombine in nature.     

In vitro synthesis of structural and nonstructural proteins of Sendai and SV5 viruses

The messenger RNAs of SV5 and two strains of Sendai virus were isolated from infected cells and translated in a wheat germ cell-free system. Comparison of the peptide maps of the polypeptides synthesizedin vivo andin vitro established that the nonglycosylated polypeptides P, NP, and M of both SV5 and Sendai virus had been synthesizedin vitro. In immunoprecipitation studies of the putative SV5 polypeptides synthesizedin vitro, antiserum against whole virions precipitated NP, P, and M, and other polypeptides which did not comigrate with mature virion polypeptides. Monospecific antisera against the HN and F glycoproteins precipitated two of the latter polypeptides with molecular weights of ～55,000 and 50,000, respectively, suggesting that the nonglycosylated forms of these polypeptides had been synthesizedin vitro. Polypeptide C, previously found in Sendai virus-infected cells and proposed to be a virus-specific nonstructural polypeptide, has been found to exhibit strain-specific differences in migration in polyacrylamide gels. Polypeptides with the appropriate strain-specific migration have been synthesizedin vitro with mRNAs from cells infected with the different Sendai virus strains, and shown by peptide mapping to be the same polypeptides as those synthesizedin vivo. The results have thus provided further evidence that C is a virus-coded nonstructural protein. Another polypeptide (C′), with migrates slightly slower than C, has been found in infected cells and synthesizedin vitro. This polypeptide also exhibits strain-specific differences in migration in polyacrylamide gel electrophoresis, and has been shown by peptide mapping to be similar to C. The explanation for the difference in migration between C and C′ is as yet unknown.     

Specificity among barley yellow dwarf viruses in enzyme immunosorbent assays

Parallel homologous and heterologous enzyme-linked immunosorbent assays revealed a marked homologous specificity among four isolates of barley yellow dwarf virus (BYDV). Relatively weak heterologous reactions permitted division of the viruses into two groups. The RPV and RMV isolates have some common antigens, but appear to be unrelated to the MAV and PAV isolates, which are related to each other. A fifth BYDV isolate, SGV, did not react strongly with any of the four virus-specific globulins studied, but faint reactions suggested a relationship with PAV.     

Characterization of inducible type-C RNA viruses of mouse strains from different geographic areas

The distribution of endogenous type-C RNA viruses was studied in inbred strains of mice and some subspecies of Mus musculus of different geographical origins. The following groups of inducible viruses were characterized by their host range and immunological properties: (1) viruses indistinguishable from one of the three prototype viruses endogenous to BALB/c mice; (2) viruses coding for proteins immunologically related to different prototype endogenous viruses; (3) viruses whose p12 structural proteins were immunologically indistinguishable from that of BALB:virus-2, but whose p30 major structural proteins and envelope glycoproteins differed immunologically from those of previously characterized endogenous viruses. These findings suggest that endogenous viruses have undergone numerous genetic interactions during the process of evolution leading to inducible viruses of present day mouse strains. A class of xenotropic virus spontaneously released by NZB mice is endogenous to but not inducible from embryo cells of other previously studied mouse strains. Viruses which could not be distinguished from the NZB xenotropic virus by host range analysis or radioimmunological techniques were chemically inducible from embryo cells of several mouse strains originating in Asia and Europe. These results indicate that the biological regulatory mechanisms that affect expression of this virus have evolved differently in such strains from control mechanisms that developed in standard inbred strains.     

Assessment of the antigenic relatedness among H3 hemagglutinins of type A influenza viruses by competition radioimmunoassay.

Competition radioimmunoprecipitation assays were used to assess the antigenic relatedness of the hemagglutinins of four of the major variants of type A influenza viruses of the H3N2 subtype. Purified whole virus preparations representative of A/Hong Kong/68 (A/HK/68), A/England/72 (A/Eng/72), A/Port Chalmers/73 (A/PC/73), and A/Victoria/75 strains were used as competing antigens. These antigens were used to inhibit the binding of iodinated preparations of purified HA obtained from each of the variants to their homologous antiserum. These analyses indicated that the degree of cross-reactivity of the three later variants with A/HK/68 was inversely related to the sequence of their isolation from human populations and that each of these variants differed antigenically from the immediately preceding and/or succeeding variants by approximately 50%. A fifth strain A/Scotland/74 (A/Scot/74) was also used as a competing antigen and it did not follow the chronological pattern observed with the four major variants. The major proportion of the cross-reactivity observed between these strains appears to be mediated through antibodies with lower avidity for heterologous HAs. However, heterologous competition assays identified two identical antigenic determinants; one was expressed on the A/HK/68, A/Eng/72, A/PC/73, and A/Scot/74 variants and the other was found on only the latter three.     

Immunological evidence for the synthesis of all canine distemper virus polypeptides in chronic neurological diseases in dogs. Chronic distemper and old dog encephalitis differ from SSPE in man.

Immunoprecipitation of the polypeptides of canine distemper virus (CDV) from lysates of infected cells has revealed that the serum and cerebrospinal fluid (CSF) of dogs with chronic distemper and old dog encephalitis contain high levels of antibody to all the viral structural polypeptides, indicating that all of these polypeptides are being synthesized in the dogs. These findings, and the fact that infectious virus has been isolated from explants of brain tissue without cocultivation techniques, differ from those with measles virus in subacute sclerosing panencephalitis (SSPE) in which there is a lack of antibody to the virus membrane (M) protein and cocultivation of brain explants with permissive cells is required for virus isolation. These results indicate that CDV undergoes complete replication in the brain in the persistent infection resulting in chronic neurological disease, in contrast to the situation with measles virus in SSPE in which replication appears to be abortive.     

Structural homology of the major internal proteins of endogeneous type C viruses of two distantly related species of Old World monkeys: Macaca arctoides and Colobus polykomos

The major internal protein (p30) of MAC-1, an endogenous type C virus of Macaca arctoides and the p30 of CPC-1, an endogenous type C virus of Colobus polykomos were purified and subjected to primary structure analysis. Despite the distant evolutionary relationship (≈20 Myr) between these two species of Old World monkeys, the amino acid compositions of the viral p30s were very similar and their COOH-terminal sequences (5 residues) were found to be identical. Moreover, the NH₂-terminal sequences (up to 36 residues) differed only in three positions. Both the NH₂- and COOH-terminal sequences showed extensive homology to respective sequences of p30s of known type C viruses from other mammalian species. Statistical analysis of the sequence relatedness revealed that MAC-1 and CPC-1 p30s are not more closely related to the p30s of another family of endogenous primate viruses isolated from baboons (Papio genus) than they are to p30s of those viruses isolated from lower mammals. On the basis of these p30 sequence relationships MAC-1 and CPC-1 together with MMC-1, an endogenous virus of Macaca mulatta, can be classified into a new (fourth) subgroup. Avian reticuloendotheliosis virus and its relatives, transforming viruses of birds are more related to this primate virus subgroup than to any of the three other subgroups of mammalian type C viruses.     

Correlation of pathogenicity and gene constellation of influenza A viruses. II. Highly neurovirulent recombinants derived from non-neurovirulent or weakly neurovirulent parent virus strains.

Mixed infections with various nonneurovirulent or weakly neurovirulent influenza A strains yielded recombinants that were highly neurovirulent for mice. A correlation was detected between gene constellation and neurovirulence of these recombinants. For the recombination pair FPV/A2-England the Pol 1 and Ptra genes had to be derived from the human strain; while the HA and/or M gene has to be from FPV in order to obtain highly neurovirulent recombinants. For the FPV/PR8 pair only the Pol 1 gene needs to be derived from PR8 while the HA gene had to be from FPV in order to get highly neurovirulent recombinants. The derivation of the other genes does not appear to be important in this respect. Recombinants between FPV and the A2-Singapore influenza strain do not exhibit significant neurovirulence suggesting that at least one parent strain should be adapted to grow in mice in order to obtain neurovirulent recombinants. In addition, there is a correlation between pneumovirulence and growth in mouse kidney cells, but neurovirulence for mice and pathogenicity for chickens was not correlated. The data presented here demonstrate in principle the possiblity of an increase in pathogenicity in recombinants derived from less or nonpathogenic parent viruses.     

Formation of the guanylylated and methylated 5′-terminus of vaccinia virus mRNA

mRNA was synthesized in vitro by vaccinia virus particles in the presence of S-adenosyl-[methyl-³H]methionine and α- or β,γ-³²P-labeled ATP or GTP. The two 5′-terminal structures m⁷G(5′)ppp(5′)G^m and m⁷G(5′)ppp(5′)A^m were isolated after P₁ nuclease digestion of the RNA. The distribution of radioactivity between the methylated mononucleotides and inorganic phosphate released by nucleotide pyrophosphatase treatment of m⁷G(5′)ppp(5′)G^m and m⁷G(5′)ppp(5′)A^m was consistent with the formation of the latter structures by condensation of pG residues from GTP with ppG- and ppA- at the 5′-termini of vaccinia mRNAs. An alternative mechanism involving the transfer of ppG residues to pA- at the 5′-terminus of the RNA was ruled out by the ³²P-labeling data as well as by the formation of m⁷G(5′)ppp(5′)A^m- when β,γ-methylene-GTP was substituted for GTP. At limiting concentrations of S-adenosylmethionine, the predominant methylated 5′-terminal structure was m⁷G(5′)ppp(5′)N- in which the penultimate nucleoside was unmethylated; in the absence of S-adenosylmethionine, G(5′)ppp(5′)N- as well as unblocked ppN- termini were detected. On the basis of the structures formed under various conditions, the following sequence of reactions was considered:

${}^{\mathrm{\gamma \beta \alpha }}\text{ppG}\text{+}{}^{\mathrm{\beta \prime \alpha \prime }}\text{ppN}\text{?→}\text{G}\text{(5′)}\text{N}\text{?+}{}^{\mathrm{\gamma \beta }}\text{PPi}$

$\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoMet}\text{→}\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoHcy}$

$\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}\text{?+}\text{AdoMet}\text{→}\text{m}{}^7\text{G}\text{(5′)}\text{ppp}\text{(5′)}\text{N}{}^{\mathrm{m}}\text{+}\text{AdoHcy}$

     

Inhibition of vaccinia virus late protein synthesis by isatin-beta-thiosemicarbazone: characterization and in vitro translation of viral mRNA.

Thespecific effect of istin-βthiosemicarbozone (IBT) was manifested after vaccinia virus late protein synthesis had commenced. At 6 hr after infection, viral protein synthesis was inhibited by about 9596. We confirmed that λ portion of the virus-specific RNA appears to be degraded (B. Woodson and W. K. Joklik, 1965, Proc. Nat. Acad. Sci. USA 54,946–953). Nevertheless, the amount of viral RNA that was capped, properly methylated, and polyadenylylated, was reduced by only about 50%. Moreover, RNA from IBT-treated cells stimulated cell-free protein synthesis to one-half the level obtained with RNA from control cells. Polyacrylamide gel electrophoretic analysis further demonstrated that RNA from IBT-treated cells was translated into late viral proteins in vitro. Thus, it seems possible that the inhibition of protein synthesis in IBT-treated cells does not result entirely or directly from either an inhibition of mRNA synthesis or from λ depletion of mRNA caused by accelerated degradation. An alternative possibility, that accelerated degradation is secondary to λ more immediate effect of the drug on protein synthesis, was considered.     

Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus

In search of a molecular basis underlying the variations in virulence observed with different strains of Newcastle disease virus, a comparative study has been carried out on the biosynthesis and function of the viral glycoproteins. Five virulent (Italien, Herts, Field Pheasant, Texas, Warwick) and five avirulent strains (La Sota, B₁, F, Queensland, Ulster) have been analyzed. They were grown in five different host systems (embryonated eggs, cultures of BHK21-F, MDBK, chick embryo, and chick chorioallantoic membrane cells).Glycoprotein F (MW 56,000) which is responsible for hemolysis and cell fusion has been found with all strains to be derived by proteolytic cleavage from the precursor glycoprotein F_o (MW 68,000). With strains Queensland and Ulster, in addition, a precursor glycoprotein HN_o (MW 82,000) has been identified which is converted, again by proteolytic cleavage, into the hemagglutinin-neuraminidase glycoprotein HN (MW 74,000). Cleavage of F_o is necessary for the expression of cell fusing and hemolytic activity, and the available evidence suggests that cleavage of HN_o is paralleled by an enhancement of hemagglutinating and neuraminidase activity. However, activation of the glycoproteins is not required for virus assembly. Thus, virus particles containing the precursor F_o may be formed which have a reduced infectivity. Infectivity is even lower, if both glycoproteins are present in the uncleaved form. After in vitro treatment with trypsin, such particles display full biological activity.Whether the glycoproteins are cleaved in vivo depends on the virus strain and on the host cell. With virulent strains, cleavage occurs in all host systems analyzed, and the virions formed contain HN and F. With avirulent strains, however, this is the case only in the embryonated egg and in cultures of chorioallantoic membrane cells. All other cells produce particles containing uncleaved glycoproteins. From these observations the following conclusions can be drawn: only a few host systems are permissive for avirulent strains, i.e., they produce highly infectious virus; other systems are nonpermissive for these strains, i.e., they produce defective virus; in contrast, all host systems studied are permissive for virulent strains. This concept is supported by the finding that multiple replication cycles and plaque formation occur only in permissive cells or in a nonpermissive culture after substitution of trypsin. Thus, plaque assays are now available for avirulent strains, either by the use of MDBK and chick embryo cells in the presence of trypsin or by the use of chorioallantoic membrane cells. These observations demonstrate striking differences in host range between virulent and avirulent strains which are determined by the susceptibility of the envelope glycoproteins to proteolytic cleavage. It is suggested that these differences account at least in part for the variations in the virulence of Newcastle disease virus.     

Serological analysis of 17 baculoviruses from subgroups A and B using protein blot immunoassay

We used the protein blot radioimmunoassay technique (Smith and Summers, 1981) to evaluate the immunoreactivity of antisera made against SDS-disrupted baculovirus structural proteins. Eight antisera (six for NPVs and two for GVs) were tested against eleven nuclear polyhedrosis (NPV) and six granulosis viruses (GV). Homologous reactions revealed that SDS-disrupted baculovirus antigens elicited antibodies which reacted with several of the virus proteins. Heterologous reactions of each viral antiserum were determined with a total of 17 baculoviruses. Using these data and that of Smith and Summers (1981), we were able to detect a number of shared antigenic determinants which revealed some specific serological relationships among some of the 17 viruses tested. The MNPVs showing distinct serological relatedness included those of Autographa californica, Rachiplusia ou, Anticarsia gemmatalis, Choristoneura fumiferana, and Orgyia pseudotsugata. Heliothis armiger MNPV and Heliothis zea SNPV shared cross-reacting antigenic determinants as did Trichoplusia ni SNPV, Pseudoplusia includens SNPV, and Heliothis zea SNPV. The GVs. of T. ni, H. armiger, and Spodoptera frugiperda were also serologically related. Also reactions were detected which indicated the presence of common antigenic determinants among different baculovirus subgroups in addition to those originally detected in our earlier study (Smith and Summers, 1981). Because of the large number of structural proteins in baculoviruses and the lack of information concerning their structural or functional roles in the virus group specific antigenic determinants could not be identified. However, protein blot RIA allows specific identification of baculoviruses and the number and molecular weight of homologous and heterologous proteins sharing antigenic determinants.     

Biological activities of monoclonal antibodies reactive with antigenic sites mapped on the G1 glycoprotein of La Crosse virus

Monoclonal antibodies have been prepared which are specific for the G1 glycoprotein of La Crosse virus. By competitive radioimmunoassay, 20 IgG-producing clones were found to map in eight antigenic sites; three distinct and five which showed individual patterns of partial competition indicating they may be in close proximity. Unique in situ trypsin cleavage sites on G1 have helped in orienting these defined epitopes relative to the viral membrane. Antibody molecules belonging to one epitope (H) mapped on the trypsin-resistant part of G1 and had negative or extremely low neutralizing and hemagglutination inhibition activities. Seven epitopes were located on the trypsin-sensitive part of G1, a 25,000-Da region which is probably the amino terminus of the protein. Antibodies binding to six of these seven epitopes (A, B, D, E, F, and G) were positive for neutralization and inhibition of hemagglutination, but exhibited a wide range of activities. Epitopes A, F, and G seem to be in an immunodominant region containing the primary site(s) for attachment to cell receptors. Antibody specific for the remaining epitope (C) was unique in that it bound to a site closely adjacent to neutralizing antibody sites, enhanced antibody binding to epitopes A and G, but lacked the capacity to neutralize viral infectivity or inhibit hemagglutination. Enhancement of antibody binding also occurred between two other closely adjacent sites (B and D) and one other distinct epitope (G). In addition, antibody from an IgM-producing clone competed with antibodies to these same four epitopes (B, C, D, and G), indicating they are in close proximity. These data have been used to construct an antigenic map that may now be used as a working model for the study of virus neutralization.