479. Development and Characterization of a New Noncytopathic Vector Derived from Semliki Forest Virus

Abstract

Top of pageAbstract Alphavirus vectors can express high levels of recombinant proteins in many different cell types. However, this expression is transient, due to the cytopathic nature of viral replication. Several noncytopathic mutants have been obtained from Sindbis virus (SIN) and Semliki Forest virus (SFV), which could be used when sustained transgene expression is desired. However, these noncytopathic vectors are generally only able to replicate in a limited number of cell lines, and have a low packaging effciency. In order generate new SFV noncytopathic vectors we have introduced mutations described to turn SIN noncytopathic into conserved positions in SFV. Interestingly, we found that one of the vectors generated in this way, which contained mutation P718T in nsp2, while apparently not able to replicate in most cells, gave rise with a relative high frequency to noncytopathic variants that formed colonies without selection when using LacZ as a reporter gene. We rationalized that these noncytopathic variants were due to second mutations that appeared naturally in this vector. To isolate these noncytopathic variants we inserted the pac gene into the SFV P718T mutant and puromycine resistant cell clones were selected in BHK cells. The noncytopathic replicons were then cloned by RT-PCR. As hypothesized, one noncytopathic replicon was rescued in this way containing in addition to P718T a second mutation in the nuclear localization signal of nsp2. This noncytopathic mutant had a lower level of replication as compared to the wild type replicon, but it was able to express heterologous proteins at similar levels. Further characterization of this double mutant will be discussed in detail.