Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus

Bovine respiratory syncytial (BRS) virus causes a severe lower respiratory tract disease in calves similar to the disease in children caused by human respiratory syncytial (HRS) virus. While there is antigenic cross-reactivity among the other major viral structural proteins, the major glycoprotein, G, of BRS virus and that of HRS virus are antigenically distinct. The G glycoprotein has been implicated as the attachment protein for HRS virus. We have carried out a molecular comparison of the glycoprotein G of BRS virus with the HRS virus counterparts. cDNA clones corresponding to the BRS virus G glycoprotein mRNA were isolated and analyzed by dideoxynucleotide sequencing. The BRS virus G mRNA contained 838 nucleotides exclusive of poly(A) and had a major open reading frame coding for a polypeptide of 257 amino acid residues. The deduced amino acid sequence of the BRS virus G polypeptide showed only 29 to 30% amino acid identity with the G protein of either the subgroup A or B HRS virus. However, despite this low level of identity, there were strong similarities in the predicted hydropathy profiles of the BRS virus and HRS virus G proteins. A cDNA molecule containing the complete BRS virus G major open reading frame was inserted into the thymidine kinase gene of vaccinia virus by homologous recombination, and a recombinant virus containing the BRS virus G protein gene was isolated. This recombinant virus expressed the BRS virus G protein, as demonstrated by Western immunoblot analysis and immunofluorescence of infected cells. The BRS virus G protein expressed from the recombinant vector was transported to and expressed on the surface of infected cells. Antisera to the BRS virus G protein made by using the recombinant vector to immunize animals recognized the BRS virus attachment protein but not the HRS virus G protein and vice versa, confirming the lack of antigenic cross-reactivity between the BRS and HRS virus attachment proteins. On the basis of the data presented here, we conclude that BRS virus should be classified within the genus Pneumovirus in a group separate from HRS virus and that it is no more closely related to HRS virus subgroup A than it is to HRS virus subgroup B.     

Differential accumulation of potato tuber mRNAs during the hypersensitive response induced by arachidonic acid elicitor

Accumulation of messenger RNAs in potato tuber discs was analysed during the hypersensitive response induced by treatment with the biotic elicitor arachidonic acid. In vitro translation of polysomal poly(A)⁺ RNAs indicated that the accumulation of some sixteen mRNAs varied following treatment with arachidonic acid, and that the level of thirteen of these was increased. Two cDNA closes (pSTH-1 and-2) were isolated from a library of elicitor-treated tissue cDNAs. Northern blot analysis using these clones as molecular probes indicated that the levels of at least two mRNAs were markedly increased after elicitor treatment. In hybrid-released translation experiments, each of the cDNA clones selected more than one mRNA. Translation of these mRNAs yielded two polypeptides of M_r 45 000 (for the pSTH-1 clone), and three polypeptides of M_e 17 000 (for the pSTH-2 clone). The low molecular weight polypeptides may correspond to potato pathogenesis-related (PR) proteins.     

cDNA cloning of six mRNAs induced by TMV infection of tobacco and a characterization of their translation products

A cDNA library was constructed to 10-15 S poly(A) RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco. By differential colony hybridization of 1400 transformants, 32 clones were obtained corresponding to TMV-inducible tobacco mRNAs. These clones were subdivided into six clusters on the basis of cross-hybridization of the inserts. By Northern blot hybridization it was shown that three of the corresponding mRNAs were strongly induced by spraying tobacco plants with salicylic acid, whereas one mRNA was weakly induced by this treatment. All mRNAs were systemically induced in plants in which only the lower leaves were locally infected by TMV. Hybrid-selected translation was performed, using six clones representing one cluster each, followed by immunoprecipitation using an antiserum to purified pathogenesis-related (PR) proteins. Four clones yielded precipitable translation products. One of these clones represented a cluster of PR-1 clones, another clone encoded the thaumatin-like (TL) protein of tobacco which may correspond to PR-P or −Q.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.     

Production and use of cDNA clones from arabis mosaic virus

Arabis mosaic virus (AMV) genomic RNAs were converted to dsDNA and cloned into bacterial plasmids. Insert sizes of cDNA clones ranged from 0·2 to 3·2 kbp. Restriction enzyme mapping identified clones representing at least 90% of the RNA-2 genome. A 0·9 kbp clone specific to RNA-1 was also identified. Northern blot hybridisations of AMV RNAs with clones from either RNA-1 or RNA-2 showed no cross reactions. The sensitivity of virus detection in dot hybridisation was 15 pg of purified genomic RNA and 40 pg of purified virus particles. The possibility of using cDNA clones for the detection of AMV in strawberry sap was demonstrated. Two AMV dsRNAs corresponding to genomic RNAs in size were isolated from infected cucumber plants and reacted in hybridisation experiments.     

The genome of respiratory syncytial virus is a negative-stranded RNA that codes for at least seven mRNA species.

The RNA from purified respiratory syncytial (RS) virions and the RNAs from RS virus-infected cells were isolated and characterized. The RNA from RS virions was found to be a unique species of single-stranded RNA of approximately 5 x 10(6) daltons. Specific annealing experiments demonstrated that at least 93% of the virion RNA was of negative (nonmessage) polarity. Eight major and three minor species of virus-specific RNA were detected in the cytoplasm of RS virus-infected HEp-2 cells. The largest intracellular RNA species comigrated with RNA from purified virions, was not polyadenylated, and was synthesized only in the presence of concomitant protein synthesis. The seven major smaller species of RNA were synthesized in the presence of an inhibitor of protein synthesis. These RNAs were all polyadenylated and were shown to be RS virus specific by their ability to anneal specifically to purified virion RNA. The sum of the sizes of the major RS virus-specific polyadenylated RNAs was sufficient to account for the coding capacity of the RS virus genome (within the limits of reliability of the methods we have used to determine size).     

Characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus JHM.

The intracerebral inoculation of Lewis rats with the murine coronavirus MHV-JHM leads in the majority of animals to acute encephalitis and death within 14 days. Viral RNAs isolated from the brains of animals 5 to 7 days after infection were compared by Northern blot analysis with the RNAs produced during the lytic infection of Sac(-) or DBT cells with wild-type MHV-JHM (wt virus). Reproducibly, the subgenomic mRNAs 2 and 3 but no other viral RNAs were significantly larger in the brain-derived material. All viruses isolated from infected brain material displayed and maintained this altered mRNA profile when cultivated in Sac(-) or DBT cells. A virus isolated from the infected brain material, MHV-JHM clone 2 (cl-2 virus), has been further characterized. This isolate grew in tissue culture and induced cytopathic effects comparable to those induced by wt virus. However, the mRNAs 2 and 3 produced in cl-2 virus-infected cells had molecular weights ca. 150,000 larger than those produced in cells infected with wt virus. There was no detectable difference in genome-sized RNA (mRNA 1) or subgenomic mRNAs 4, 5, 6, and 7 as determined by electrophoresis in agarose gels. T1-resistant oligonucleotide analysis of genomic RNA revealed one additional and one missing oligonucleotide in the fingerprint of cl-2 virus compared with wt virus. The oligonucleotide fingerprints of intracellular mRNA 3 were identical for both viruses. Pulse-labeling with [35S]methionine in the presence of tunicamycin showed that the primary translation product of mRNA 3, the E2 apoprotein, was ca. 15,000 larger in molecular weight in cl-2 virus-infected cells. These data show that viruses with larger mRNAs 2 and 3 (the latter encoding an altered E2 glycoprotein) are selected for multiplication in rat brains. Mechanisms for the generation of such variants and the possible nature of their selective advantage are considered.     

Characterization and cloning of rat dorsal prostate mRNAs. Androgen regulation of two closely related abundant mRNAs

Electrophoresis of rat dorsal prostate mRNAs on agarose gels containing methyl mercury hydroxide indicates the presence of several highly abundant mRNAs. In vitro translation of the total mRNAs in a cell-free system, followed by polyacrylamide gel electrophoresis, yields protein products including two intense bands corresponding to 23,000 and 21,000 Da. Following castration of rats, these in vitro translation products of dorsal prostate mRNAs are absent. However, the dorsal prostate levels of these two proteins are returned to normal in castrated rats which have received testosterone. In order to investigate these abundant mRNAs of the dorsal prostate, we have constructed double-stranded cDNA clones using poly(A+) RNA extracted from that rat tissue. Clones containing sequences complementary to abundant mRNAs were selected kinetically by colony hybridization with 32P-labeled dorsal prostate cDNA. Further characterization of individual clones was accomplished by restriction mapping and Northern blot analysis. One clone, pM-40, was found to be near full length and was used for further studies. Interestingly, in hybrid-arrested cell-free translation, clone pM-40 completely arrests the translation of both the 23,000- and 21,000-Da protein products indicating close sequence homology between these two proteins. Furthermore, dot hybridization experiments demonstrate that, in the dorsal prostate, the pM-40-specific mRNAs decrease following castration and are restored by testosterone administration. However, the low levels of the same mRNAs in the ventral prostate are not altered by androgen manipulation. Thus, two closely related, androgen-dependent tissues maintain differential regulation of the pM-40 gene(s). This system provides an opportunity to study in two tissues the differential regulation of a gene that may be duplicated or that may code for two separate proteins.     

Antigen mimicry involving measles virus hemagglutinin and human respiratory syncytial virus nucleoprotein.

Intergenic antigenic relationships between measles virus and respiratory syncytial (RS) virus-specific structural components were studied by using monoclonal antibodies. Of 75 monoclonal antibodies against these components, only one, an anti-measles virus hemagglutinin monoclonal antibody, cross-reacted. Immunofluorescence analysis of measles virus- and RS virus-infected cells with this monoclonal antibody showed qualitatively different staining patterns which indicated that the antigen involved in cross-reaction was the RS virus nucleoprotein or phosphoprotein. A radioimmunoprecipitation assay showed the antigen to be the nucleoprotein.     

At least 104 nucleotides are transposed from the 5' terminus of the avian sarcoma virus genome to the 5' termini of smaller viral mRNAs.

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3' half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA(cDNA5') that is complementary to a sequence of 101 nucleotides found at the 5' terminus of the ASV genome, but not within the 3' half of the genome. We proposed previously (Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5' termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA5' in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASVmRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a "capped" oligonucleotide identical to that found at the 5' terminus of the ASV genome. When hybridized with cDNA5', each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA5'. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5' terminus of the viral genome and had identical "fingerprints" when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3' half of the ASV genome did not form hybrids with cDNA5'. We conclude that a sequence of more than 100 nucleotides is transposed from the 5' end of the ASV genome to the 5' termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.     

Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein

A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development.     

Probes for rare mRNAs reveal distributed cell subsets in canary brain

cDNA clones of 7 low-abundance canary brain RNAs hybridize in situ to different subsets of brain cells. Although these cell sets are distinct, they are dispersed in a variety of brain regions with overlapping anatomical distributions. These cDNA clones were initially selected by their relative hybridization to forebrain and rest-of-brain RNAs and represent a sampling of a much larger population of differentially expressed RNAs present at individual concentrations of 10(-7) to 10(-4) as a fraction of polyadenylated RNA mass. Our results suggest the existence of several thousand low-abundance brain mRNAs likely to be distributed in diverse and overlapping brain cell subsets. Furthermore, our experiments define a simple and general strategy for producing and analyzing molecular probes for subsets of brain cells and provide an initial set of useful reagents for further study of brain organization and development.     

Multiple chick tropoelastin mRNAs

Several overlapping chick tropoelastin cDNAs were isolated from a lambda gt11 cDNA library constructed from whole 10 day chick embryo total RNA. Comparison of the nucleotide sequence of the 2.3 kb tropoelastin cDNA to the sequences published by Bressan et al. (1) and Tokimitsu et al. (2) revealed the presence of two inserts (72 and 30 base pairs) in the cDNA derived from embryonic tissue. Northern blot analysis of 14 day embryonic aortae RNA with tropoelastin cDNA clones showed hybridization to a 3.5 kb mRNA. However, S1 nuclease protection experiments performed on RNA extracted from the same tissue showed that at least two if not more tropoelastin mRNAs exist and that the proportion of each varies in the ages examined. These results provide an origin and substantiate the differential expression of the multiple tropoelastin polypeptides found in developing chick aortic tissue.     

The disease resistance response in pea is associated with increased levels of specific mRNAs

A cDNA library was constructed using poly(A)⁺RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using³²P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected, which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for 16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance during infection. Results of³H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation of the fungus-inducible mRNAs which hybridized with the clones.