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Abstract
Severe acute respiratory syndrome (SARS), the first world pandemic in the 21st century was caused by a novel corona virus, SARS-CoV. The purpose of this research was to investigate roles and influence of cellular aging, host cell receptor angiotensin converting enzyme 2 (ACE2), growth factor supplements, and endocytic pathways in the replication cycle of SARS-CoV. Significant step wise increases in SARS-CoV titers directly correlated with cell culture age (from 3 weeks up to 15 months), being the lowest in the 3 weeks old cell cultures and the highest in 15 months old cultures. Both ACE2 protein and mRNA levels had significant age related increases in cells continuously maintained in the culture for 3 weeks, 12 weeks, 8 months and 15 months. Higher expression of cell bound ACE2 had positive correlation with higher virus titers, but higher concentration of solubilized ACE2 in the cell culture media was associated with significant decreases in viral titers. Delivery of exogenous ACE2 using a plasmid expression vector significantly increased expression of ACE2 in the young but not in the old cells. However, decreasing ACE2 expression via specific small interfering RNAs (siRNAs) resulted in significantly lower titers in both old and young cells. To test influence of growth factor supplements, bovine pituitary extract (BPE) was used. BPE was found to be a strong inhibitor of SARS-CoV replication, decreasing titers by 400 fold. Although protein scouting revealed the presence of growth hormone (GH) in the BPE fractions with retained antiviral properties, GH alone did not provide protection. Interestingly BPE analysis revealed the presence of multiple heat shock proteins. When silenced using siRNAs, HSPA12B and UTY genes significanly decreased SARS-CoV titers, while silencing of HSPBP1, HSPB8, HSPB9, UNC45B, HSPBAP1, STIP1, UBE2Q1 and HSPA9 led to significant increases in SARS-CoV titers. Targeting clathrin heavy chain and caveolin-1 with specific siRNAs, significantly reduced viral titers, ACE2 protein and mRNA levels. Overall the results of these experiments documented the importance of two endocytic pathways, characterized cell culture age dependent - SARS-CoV replication and ACE2 expression, and discovered bidirectional roles of heat shock proteins in SARS-CoV life cycle.