ABSTRACT
The aim of this study was to determine the impact of lentiviral transduction on primary murine B cells. Studying B cell activities in vivo or using them for tolerance induction requires that the cells remain unaltered in their biological behavior except for expression of the transgene. As we show here, murine B cells can efficiently be transduced by lentiviral, VSV-G-pseudotyped vectors without the necessity of prior activation. Culture with LPS gave enhanced transduction efficiencies but led to the upregulation of CD86 and proliferation of the cells. Transduction of naive B cells by lentiviral vectors was dependent on multiplicity of infection and did not lead to a concomitant activation. Furthermore, the transduced cells could be used for studies in the NOD mouse system without altering the onset of diabetes. We conclude that lentiviral gene transfer into naive B cells is a powerful tool for manipulation of B cells for therapeutic applications.
Subject(s)
B-Lymphocytes/immunology , Lentivirus/genetics , Animals , Antigens, CD/biosynthesis , Antigens, CD/immunology , B-Lymphocytes/cytology , B-Lymphocytes/virology , Cell Division , Cell Line , Diabetes Mellitus, Type 1/immunology , Female , Flow Cytometry , Genetic Vectors/genetics , Green Fluorescent Proteins , Humans , Immune Tolerance/genetics , Immune Tolerance/immunology , Lentivirus/immunology , Lentivirus/pathogenicity , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred NOD , Spleen/cytology , Transduction, Genetic/methodsABSTRACT
The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F)(265) is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.
Subject(s)
Glycoproteins/metabolism , Lymphocytic choriomeningitis virus/metabolism , Proprotein Convertases , Serine Endopeptidases/metabolism , Viral Proteins/metabolism , Animals , CHO Cells , Cell Line , Chlorocebus aethiops , Cricetinae , Genetic Vectors , Glycoproteins/chemistry , Glycoproteins/genetics , HeLa Cells , Humans , Lymphocytic choriomeningitis virus/genetics , Retroviridae/genetics , Vero Cells , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
Lymphocytic choriomeningitis virus (LCMV) is a noncytopathic arenavirus shown to infect a broad range of different cell types. Here, we combined the beneficial characteristics of the LCMV glycoprotein (LCMV-GP) and those of retroviral vectors to generate a new, safe, and efficient gene transfer system. These LCMV-GP pseudotypes were systematically compared with vectors containing the widely used amphotropic murine leukemia virus envelope (A-MLVenv) or the vesicular stomatitis virus G protein (VSV-G). Production of LCMV-GP-pseudotyped oncoretroviral and lentiviral vectors by transient transfection resulted in vector titers similar to those with A-MLVenv or VSV-G. In contrast to A-MLVenv particles, LCMV-GP pseudotypes could be efficiently concentrated by ultracentrifugation without loss of vector titer. Unlike the cell-toxic VSV-G, a stable retroviral packaging cell line constitutively expressing LCMV-GP could be established. Vectors pseudotyped with LCMV-GP efficiently transduced many cell lines from different species and tissues relevant for gene therapy. Transduction of human glioma cells was studied in detail. These cells are a major target for cancer gene therapy and were transduced more efficiently with LCMV-GP-pseudotyped vectors than with the generally used A-MLVenv particles. The high stability, low toxicity, and broad host range make LCMV-GP-pseudotyped vectors attractive for gene transfer applications. The recombinant LCMV-GP-pseudotyped vectors will also allow functional characterization of naturally occurring and recombinant LCMV-GP variants.
