Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.     

Characterization of ribosome binding on Rous sarcoma virus RNA in vitro.

We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5'-proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins.     

Characterization of messenger RNA by direct translation from agarose gels

A method for characterizing nanogram quantities of poly(A)-containing messenger RNAs that have been fractionated according to size by electrophoresis through agarose gels has been developed. The mRNAs from Friend leukemia cells were identified by the protein products they encode, as determined by slicing the agarose gel and directly translating the enclosed mRNA with an extract from rabbit reticulocytes that had been treated with micrococcal nuclease. A number of parameters which affect the efficiency of translation in this system have been examined. These include the sensitivity of the in vitro translational system to RNA, the agarose concentration, the incubation temperature, and the addition of either exogeneous tRNA or RNasin. The procedure is rapid, simple, reproducible, and applicable for the fractionation and characterization of mRNAs from any source.     

The recognition specificity of a murine helper T cell for hemagglutinin of influenza virus A/PR/8/34

Human large granular lymphocytes (LGL), which are known to be responsible for natural killer (NK) cell activity, also produced a variety of lymphokines including interleukin 2 (IL 2), colony stimulating factor (CSF), and interferon (IFN) in response to phytohemagglutinin (PHA) or concanavalin A (Con A). Human peripheral blood LGL, which were purified by removal of monocytes adhering to plastic flasks and nylon columns, followed by separation on a discontinuous Percoll gradient, and additional treatment with anti-OKT3 and Leu-M1 plus complement, were more potent producers of these lymphokines than unseparated mononuclear cells (MNC), nylon column-eluted cells, or purified T lymphocytes. Moreover, IL 2 production by LGL could be further distinguished in that it was not enhanced by the addition of macrophages or macrophage-derived factor, i.e., IL 1, whereas addition of macrophages did potentiate IL 2 production by T lymphocytes. Further analysis of cells in the LGL population using various monoclonal antibodies revealed that removal of cells with OKT11 or AF-10, a monoclonal antibody against human HLA-DR antigen, decreased IL 2 production, whereas removal of OKT8+, OKM1+, Leu-M1+, or Leu-7+ cells led to enhanced IL 2 production. The LGL population is therefore heterogeneous and includes at least three functionally and phenotypically distinct subsets. An atypical T cell subset (OKT3-, Leu-1-, OKT11+) rather than the myeloid subset of LGL (Leu-M1+ or OKMI+) was the source of LGL-derived IL 2, whereas the latter subset and/or another subset of OKT8+ cells appear to regulate this IL 2 production. In addition to performing NK activity, LGL on a per cell basis seem to be more effective than T lymphocytes in producing lymphokines, namely, IL2, CSF, and IFN.     

A mutation in the short 5''-proximal open reading frame on Rous sarcoma virus RNA alters virus production.

The 5'-proximal open reading frame on Rous sarcoma virus RNA encodes a seven-amino-acid peptide and is conserved in all avian sarcoma-leukosis retroviruses. Ribosome-binding site analysis in intact chick cells showed that the 5'-proximal AUG codon is a strong site for initiation of translation in vivo. Removal of the 5'-proximal AUG codon by site-specific mutagenesis resulted in a virus with a reduced ability either to replicate or to transform a population of chicken embryo fibroblasts. These results establish a procedure for determining sites of ribosome binding and initiation of translation on mRNAs in intact eucaryotic cells and strongly suggest that the 5'-proximal open reading frame (or its AUG codon) on Rous sarcoma virus RNA has an important role in regulating viral activity.     

Production of an extracellular chitinolytic system byTalaromyces emersonii CBS 814.70.

Summary The thermophilic fungusTalaromyces emersonii CBS 814.70 produces a thermostable extracellular chitinolytic system when cultured on chitin containing media. The chitinolytic system consists of chitinase (EC 3.2.1.14) and N-acetylglucosaminidase (EC 3.2.1.30). Using fluorescent substrate analogues, in zymogram staining of polyacrylamide gradient and isoelectric focusing gels on which the chitinase system was electrophoresed and focused, respectively, it was found that a number of bands could be resolved. Using isoelectric focusing it was observed that at least 4 extracellular forms of chitinase activity are produced.