Unveiling molecular secrets: Analysis of stable lentiviral packaging cell lines enables identification of novel viral gene functions.

Lentiviral vectors (LVVs) are widely used in gene therapy due to their ability to infect both dividing and non-dividing cells. For LVV production, the creation of stable packaging cell lines with integrated genes necessary for viral replication offer a more consistent and scalable alternative to transient plasmid transfection approach. Although the development of such stable LVV packaging cell lines has been reported, the molecular changes induced by stable and inducible viral gene expression and the impact of genome integrated viral genes on cellular pathways remain poorly characterized. For better insight, we investigated the molecular characteristics of a stable LVV packaging cell line and its host cell line (HEK293T/17) by comparing differential expressed genes. This pathway analysis revealed significant changes in pathway usage between packaging and host cell lines, influenced by different viral transgenes. Gag-pol expression was found to suppress host translational machinery, while rev and VSV-G expression modulated mitochondrial pathways, including oxidative phosphorylation. HIV-1 tat expression, on the other hand, activated histone-related genes. These regulatory shifts suggest a strategic reprogramming of host cellular states to favor viral replication, curbing protein synthesis and energy production to levels that support viral assembly but impair the host's immune defense and the production of immune-related proteins. Our findings provide a deeper understanding of the molecular changes associated with stable viral gene expression, which can inform the optimization of LVV production in gene therapy applications.