(623bt) Integrating Functional Genomics Tools to Survey Retrovirus Production In Human Cells

Retrovirus derived particles found valuable biotechnological application in Vaccinology and Gene

therapy, although challenging from the manufacturing view-point, due to the low titers and high content of

contaminant defective particles. Additionally, retroviruses such as HIV are a major world-wide health

concern with the cell-virus interaction dynamics poorly understood. Thus, an integrated overview on the

cellular determinants of retrovirus production in human cells can provide a valuable starting point and a

potential tool to address these and other issues.

In this work, we analyzed transcriptome changes between retrovirus producers and the corresponding

parental cells using two different human cell lines, and took a pathway analysis approach to identify

biochemical networks overrepresented in the virus production state. Nearly 200 pathways were identified,

majorly represented by signaling and metabolism. Retrovirus production induced transcriptome changes

associated with apoptotic and cell survival pathways suggesting a balance between both networks, a typical

feature of virus infection scenario. Fatty acid metabolism was among the most prominently up regulated

pathways, along with sphingolipid and phospholipid metabolism, suggesting increased membrane recycling

dynamics for viral replication. Amino acid degradation was notably up-regulated as well. Virus production

appears to result in higher energy demands, evidenced by the strong up-regulation of the oxidative

phosphorylation and electron transport chain. Oxidative stress, pyrimidine metabolism and protein

synthesis and post-translation modification were also highlighted.

Based on the pathway analysis output, we investigated particular transcriptome-fluxome correlations.

Amino acid catabolism profile, one of the most significantly enriched pathways in terms of gene

expression, was analyzed by HPLC and found to be considerable increased, up to 2 fold, in virus producer

cells. Other suggested pathways are currently under study, some of which further substantiate pathways

analysis results, including oxidative stress metabolism and lipid biosynthesis.

In parallel, the integration of transcriptomic and metabolomic data is being used to feed a genomescale

constraint-based reconstruction of the metabolic network, in which a lumped reaction comprising all

building blocks for virus production was included. Such flux-balance analysis based model should help to

elucidate the cell fluxome alterations induced by a virus productive state and the rules to either enforce or

inhibit such state. This work has a direct application in the field of retrovirus manufacture for Vaccinology

and Gene Therapy, providing a framework to guide the rational design of process engineering and cell line

development. It can also be relevant basic virology providing further insights on the cell-virus interaction

and disclosing potential therapeutic targets to inhibit viral replication.