RESULTS:
1 - 2 of 2 for "Jelena Andrejeva"
Single-cycle parainfluenza virus type 5 vectors for producing recombinant proteins, including a humanized anti-V5 tag antibody
Parainfluenza virus type 5 (PIV5) can cause either persistent or acute/lytic infections in a wide range of mammalian tissue culture cells. Here we have generated PIV5 fusion (F)-expressing helper cell lines that support the replication of F-deleted viruses. As proof of the principle that F-deleted single-cycle infectious viruses can be used as safe and efficient expression vectors we have cloned and expressed a humanized (Hu) version of the mouse anti-V5 tag antibody (clone SV5-Pk1). We show that multiple different cell lines can be infected and express high levels of the Hu anti-V5 antibody with Chinese hamster ovary cells expressing 20–50 mg l−1 after 5 days when cells were grown to a density of ~1×106 cells per millilitre at the time of infection. We suggest that PIV5-based vectors may be further developed to produce recombinant proteins both in vitro and in vivo.
Understanding virus resistance due to ISG15-loss-of-function
Viral infections induce profound cellular responses resulting the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity while others regulate the cellular response. For most viruses the specific antiviral ISG(s) is not known which has potential consequences for the quest for new therapeutics. The ubiquitin-like protein ISG15 is a major regulator of antiviral response and inherited ISG15-deficiency leads to autoinflammatory interferonopathies where patients exhibit elevated ISG expression in the absence of infection. Using CRISPR/Cas9 knockout technology we have recapitulated these effects in cultured cells confirming ‘free’ ISG15’s role as a central regulator of type-I IFN antiviral response. We also show that during an antiviral response ISG15-deficiency leads to significant physiological defects (inhibition of translation and proliferation) and resistance to parainfluenza viruses. We asked if virus resistance was due to the direct antiviral activity of ISGs or whether cells were non-permissive due to physiological defects. We took advantage of the knowledge that IFIT1 is the principle antiviral ISG for parainfluenza virus 5 (PIV5). Knockdown of IFIT1 restored PIV5 infection in ISG15-deficient cells confirming that resistance was due to the antiviral response and not due to physiological state related to ISG15-deficiency. We also compared infections with related viruses where IFIT1 has known intermediate antiviral activity (PIV2) and low activity (PIV3); restoration of replication with these viruses reflected their sensitivity to IFIT1 restriction. Based on the observations in IFIT1-knockdown cells we propose a novel platform for the identification of antiviral ISGs based on recovery of virus infection.