An improved in vitro and in vivo Sindbis virus expression system through host and virus engineering

Abstract

The Sindbis viral expression system enables the rapid production of high levels of recombinant protein in mammalian cells; however, this expression is typically limited to transient production due to the cytotoxicity of the virus. Limiting the lethality inherent in the Sindbis virus vector in order to enable long term, sustained expression of recombinant proteins may be possible. In this study, modifications to virus and host have been combined in order to reduce the cytopathic effects. Non-cytopathic replication competent viruses of two Sindbis viral strains, TE and 633, were developed using a non-structural protein (nsP) P726S point mutation in order to obtain persistent heterologous gene expression in infected Baby Hamster Kidney (BHK) cells and Chinese Hamster Ovary (CHO) cells. Cells infected with the P726S variant viruses were able to recover after infection, while cells infected with normal virus died within 3 days. The P726S mutation did not reduce the susceptibility of 5- and 14-day-old mice to 633 and TE viruses in vivo. In addition, animal survival with the P726S variant viruses was increased and GFP expression was sustained for at least 14 days while the 633 and TE infection resulted in short-term GFP expression or an earlier mortality. Modifications to the host BHK and CHO cells themselves were subsequently undertaken by including the anti-apoptotic gene Bcl-2 and a deletion mutant of Bcl-2 (Bcl-2Delta) as another method for limiting the cytopathic effects of the Sindbis virus. The inclusion of anti-apoptotic genes permitted higher production of heterologous GFP protein following Sindbis virus infection, and the combination of the TE-P726S virus and the CHO-Bcl-2Delta cell line showed the greatest improvement in cell survival. Sindbis virus infection also induced ER stress in mammalian cells as detected by increased PERK phosphorylation and ATF4 translation. Overexpression of Parkin, an E3 ubiquitin ligase that can protect cells against agents that induce ER stress, suppressed Sindbis virus-induced cell death in both BHK cells and in vivo studies in mice. Such findings show that viral and host modifications can improve cell survival and production of heterologous proteins, change viral behavior in vitro and in vivo, and assist in the development of new expression or gene delivery vehicles.

Figure 1

Replication and packaging competent recombinant Sindbis viruses 633, TE, 633-P726S, and TE-P726S. The 633 and TE viruses differ by an amino acid at the position 55 of the E2 protein that is Glutamine (Q) or Histidine (H). The Proline (P) to Serine (S) mutation is at the amino acid position 726 in the nsP2 domain of the nonstructural proteins. The green fluorescence protein (GFP) is expressed from the second subgenomic promoter.

Figure 2

Percentage of live (a) BHK and (b) CHO cells after infection with the Sindbis viruses 633, TE, and its P726S variants. Percentage live cells were calculated as viable cell density at each time point divided by initial viable cell density at 12 h p.i. To exclude uninfected cells, only fluorescent cells (infected cells expressing GFP) were counted (see Materials and Methods). Total recombinant GFP production of the (c) BHK and (d) CHO cells post infection. The arbitrary fluorescence unit maximum (y-axis) is set to140 for all of the fluorescence results in Fig. 2, 4, 5 for comparison purpose. (e) Western blot showing GFP expression in BHK cells at 16 hours p.i. 10 μg of total protein in the cell lysate was loaded into each lane. (f) BHK cells infected with the TE-P726S virus at 7 and 14 days p.i. and no infection control.

Figure 3

(a) Survival of the 3-day old mice after infection of the Sindbis viruses 633, TE, 633-P726S and TE-P726S. (b) Hippocampal sections of the 5-day old mice infected with the 633, 633-P726S, TE, TE-P726S viruses at 3 days and 14 days p.i. (c) Hippocampal sections of the 14-day old mice infected with the TE-P726S virus at 3 days and 14 days p.i.

Figure 4

Percentage of live (a) BHK-Bcl-2 and (b) BHK-Bcl-2Δ cells after infection with the Sindbis viruses 633, TE, 633-P726S and TE-P726S. Total GFP expression of the (c) BHK-Bcl-2 and (d) BHK-Bcl-2Δ cell lines post infection.

Figure 5

Percentage of live (a) CHO-Bcl-2 and (b) CHO-Bcl-2Δ cells after infection with the Sindbis viruses 633, TE, 633-P726S and TE-P726S. GFP expression level of the (c) CHO-Bcl-2 and (d) CHO-Bcl-2Δ cell lines post infection.

Figure 6

(a) Western blots showing PERK phosphorylation, PERK, eIF-2α phosphorylation in 293 cells that were mock infected (mock), infected with the TE virus (SV), or exposed to thapsigargin as a positive control (TG). (b) BHK cells were transfected with the firefly luciferase reporter plasmid consisting of the translational fusions of the ATF4 5' UTR prior to infection with the viruses. Luciferase activity was determined to indicate ATF4 translation level at 0 and 16 hours post-Sindbis virus infection.

Figure 7

(a) BHK cells infected with the TE12Q virus expressing Bcl-x_L (a generic inhibitor of apoptosis), Bax (pro-apoptotic), Parkin or GFP. (b) 3-day old mice infected i.c. with the TE12Q viruses expressing GFP, p35 (caspase inhibitor), and Parkin.