Characterization of retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses.
In this study, we successfully established retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope (PrM/E) proteins of each of the four serotypes of dengue viruses, which caused the most important arboviral diseases in this century. Co-sedimentation of the dengue E protein and HIV-1 core proteins by sucrose gradient analysis of the pseudotype reporter virus of dengue virus type 2, D2(HIVluc), and detection of HIV-1 core proteins by immunoprecipitation with anti-E monoclonal antibody suggested that dengue viral proteins were incorporated into the pseudotype viral particles. The infectivity in target cells, as assessed by the luciferase activity, can be inhibited by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more dengue viral sequences available from different outbreaks worldwide, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates.
Institute of Microbiology, College of Medicine, National Taiwan University, No 1 Sec1 Jen-Ai Rd, Taipei 100, Taiwan.
Hsien-Ping
HP
Hu
Szu-Chia
SC
Hsieh
Chwan-Chuen
CC
King
Wei-Kung
WK
Wang
D002460:Cell Line; D003716:Dengue Virus / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D012703:Serotyping; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D014773:Virology
D014759:Viral Envelope Proteins; C074932:prM protein, Flavivirus; D008156:Luciferases