Peter R. Brayton

Expression and function of transplantation antigens with altered or deleted cytoplasmic domains

Two mutants of the class I gene encoding the H-2Ld transplantation antigen have been constructed. In one mutant the cytoplasmic domain of the class I molecule has been altered by deletion of 24 of the 31 C-terminal residues, and in the second the C-terminal 25 residues of the cytoplasmic domain have been replaced with a unique sequence of 19 amino acids. These mutant class I genes have been transferred into mouse L cells by DNA-mediated gene transfer. Both mutant genes are expressed at normal levels on the cell surface, and they have charge properties and sizes consistent with the introduced alterations. These mutant Ld molecules can serve as target antigens for allogeneic cytotoxic T cells and as restricting elements for virus-specific cytotoxic T cells. These results show that the 24 residues replaced or deleted from the carboxy terminus of the class I molecule are not required for its transport to or integration in the plasma membrane, nor for its function as a target antigen or a restricting element during T-cell-mediated cytotoxicity.

Further characterization of mouse hepatitis virus RNA-dependent RNA polymerases.

Abstract Two temporally and enzymatically distinct RNA-dependent RNA polymerase activities associated with membranes of the mouse hepatitis virus (MHV)-infected cells have been identified previously [Brayton et al., J. Virol. 42, 847–853 (1982)]. In this paper, the subcellular distribution and functions of these two polymerases were examined. Fractionation of the postnuclear membranes by sucrose gradient sedimentation showed that the early polymerase activity (detected at 1 hr p.i.) was homogeneous, while the late polymeras e (6 hr p.i.) was associated with two distinct membrane fractions. The early polymerase synthesized a single RNA species of viral genomic size and negative sense. In contrast, the light peak of the late polymerase synthesized genomic-sized RNA of positive sense, while the heavy peak of the activity synthesized positive-sensed genomic and subgenomic mRNAs. These findings suggest that the light peak of the late polymerase represents a replication complex while the heavy peak represents a transcription complex. They also establish the essential features of the mode of replication of MHV.

Host Cell Nuclear Function and Murine Hepatitis Virus Replication

Murine hepatitis virus strains A59 and JHM replicated with equal efficiency in both nucleated and enucleated L2 cells. In addition, treatment of the host cell with either actinomycin D or alpha-amanitin, both inhibitors of host cell RNA synthesis, had no effect on virus replication. Therefore, the replication of murine hepatitis virus did not appear to depend upon either the presence of the host cell nucleus or continued host cell RNA synthesis.

Cytotoxic T lymphocytes recognize determinants on the BALB/c-H-2Ld molecule controlled by α1 and α2 but not α3 external domains

We have shown that cytotoxic T lymphocytes (CTL) raised in H-2dmice use H-2Ld but not H-2Dd or H-2Kd antigens as restricting elements in lymphocytic choriomeningitis virus (LCMV) and vesicular stomatis virus (VSV) infections. To localize the regions of H-2Ld protein recognized by CTL, we constructed a recombinant H-2Ld/Ddgene encoding a hybrid antigen with α1 and α2 external domains of H-2Ld and α3, transmembrane and cytoplasmic domains of H-2Dd. The recombinant gene was transfected into mouse cells and the hybrid molecules were characterized serologically, biochemically and functionally. In all assays, H-2Ld/Dd molecules were recognized by LCMV- and VSV-specific H-2Ld-restricted CTL in a manner similar to that of wild-type H-2Ld antigens. Analogous results were obtained with alloreactive CTL. Hybrid antigens containing the α3 domain of H-2Ld fused to α1 and α2 domains of a Qa-2,3 region-encoded antigen were not used as restricting elements by LCMV-specific CTL. These results suggest that H-2Ld-restricted CTL directed against LCMV and VSV recognize determinants controlled by the α1 and/or α2 domains of the H-2Ld molecule.

BIOLOGICAL PROPERTIES OF CLASS I MHC MOLECULES EXPRESSED AFTER DNA-MEDIATED GENE TRANSFER

ABSTRACT Mouse L cells ( H-2 k ) transfected with a genomic clone containing the gene for transplantation antigen H-2L express the H-2L d molecule on the cell surface in a form indistinguishable from the native molecules. To determine the applicability of this system to the study of major histocompatibility complex (MHC) gene function, several biological properties of a cloned H-2L d transformant, 8-5, were evaluated. When 8-5 cells were used as an immunogen in C3H ( H-2 k ) mice, an antiserum was produced with activity predominantly directed against H-2L d specificities. This illustrates the utility of this approach for defining previously uncharacterized class I gene products expressed in transformed cells. The H-2L d molecules expressed on 8-5 cells also function as direct target antigens and restricting elements for H-2L d specific and lymphocytic choriomeningitis virus specific cytotoxic T lymphocytes respectively. In both instances, recognition of 8-5 cells by effector T cells was blocked by addition of H-2L d specific antisera or monoclonal antibodies.