Infectious clone systems for filoviruses

Life Cycle Filoviruses

components of the system and the steps involved in the rescue of infectious virus. (1) Co-transfection of the plasmid carrying the full-length ZEBOV genome and the expression plasmids for the bacteriophage T7 RNA polymerase (77 Pol) and the four ZEBOV proteins associated with the ribonucleoprotein complex (L, NP, VP30, VP35); (2) - expression of the viral support proteins and the bacteriophage T7 RNA polymerase under the control of the chicken P-actin promoter; (3) - transcription of the ZEBOV genome under the control of the bacteriophage T7 RNA polymerase promoter; (4) - formation of the ribonucleoprotein complex, transcription and replication; (5) - virus maturation at the plasma membrane and subsequent budding of infectious virus particles. L RNA-dependent RNA polymerase; N nucleus; NP nucleoprotein, VP virion protein 30 and 35kDa. [altered from references #52 & #74]. • NP; ♦ VP35; O T7; A VP30; □ L

has been commonly provided by infection with a recombinant vaccinia virus [51, 55]. However, this system has the disadvantage of requiring separation of the recombinant viruses of interest from progeny of the recombinant vaccinia virus. Recently we have optimized the infectious clone system developed by Neumann and colleagues [52] to a rescuability of nearly 100% [74]. This system can now be more reliably used for the generation and analysis of mutants, particularly if rescues are unsuccessful due to incompatibility of the mutations with virus replication.

The ZEBOV infectious clone systems have been used in the past to address questions regarding the pathogenic potential of GP, which is encoded by gene 4 of the linear arranged genome (Fig. 4). In contrast to MARV, which produces only GP1i2, the predominant products of this gene for all EBOV species are the soluble secreted glycoproteins sGP and A-peptide, a small carboxyl-terminal



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