Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo.
Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.
Molecular Microbiology and Gene Therapy Unit, Institute of Medical Microbiology, University of Regensburg, 93053 Regensburg, Germany.
Diana
D
Hammer
Jens
J
Wild
Christine
C
Ludwig
Benedikt
B
Asbach
Frank
F
Notka
Ralf
R
Wagner
D016915:AIDS Vaccines / Q000276:immunology; D000409:Alanine / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics* / Q000378:metabolism; D015316:Genetic Therapy; D005998:Glycine / Q000235:genetics; D015658:HIV Infections / Q000628:therapy*; D015497:HIV-1; D006801:Humans; D051379:Mice; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes; D019444:Vaccines, DNA / Q000276:immunology; D014779:Virus Replication; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000235:genetics* / Q000378:metabolism
D016915:AIDS Vaccines; D019309:Epstein-Barr Virus Nuclear Antigens; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D054301:gag Gene Products, Human Immunodeficiency Virus; D046988:Proteasome Endopeptidase Complex; C417029:EBV-encoded nuclear antigen 1; D000409:Alanine; D005998:Glycine