(216b) Single-Cell Kinetics of Infection by An RNA Virus: a Step Toward Drug-Targeting Strategies That Resist Escape

Viruses that carry RNA genomes define some of the most challenging targets for the development of effective anti-viral drugs. Such RNA viruses, which include HIV-1, influenza A, and hepatitis C, often exhibit high mutation rates and large population sizes, enabling their adaptation to new host environments. The presence of anti-viral drugs that target specific virus functions, such as the reverse transcriptase of HIV, the neuraminidase of influenza A, or the protease of hepatitis C, select for virus escape mutants --- variant strains that are less sensitive or more resistant to the inhibitory effects of the drugs. The accumulation of multiple drug-resistance mutations in RNA viruses significantly contributes to the persistence of diverse viral diseases that include AIDS, influenza, and liver cancer. As an alternative to the more common approach of targeting single molecular functions of a virus, we are exploring more holistic approaches that acknowledge the importance of multiple interacting functions in defining the highly coordinated and potentially conserved processes of virus production within cells. Toward this end, we have employed a model RNA virus, vesicular stomatitis virus (VSV) and its infection of baby hamster kidney (BHK21) cells, to quantitatively characterize the kinetics of virus production from single cells. Our approach employs serial dilution of infected cell populations, identification of single cells by microscopy, repeated total sampling and replenishment of the infected-cell supernatant, and quantitative analysis of these samples for infectious virus progeny. Our observations highlight several key parameters for virus production from single cells: the time required for an infected cell to initiate production of virus (delay time), following this delay, the number of virus particles released per unit time (rise rate), and the total number of virus particles released by an infected cell (yield). Here we show that cell-to-cell differences in rise rates and yields for virus production vary over two orders of magnitude, while delay times are relatively constant. Our results suggest that virus-cell interactions during the earliest stages of infection are more constant, predictable and potentially targetable than interactions associated with the more variable and less predictable later stages of infection. This observation has important consequences for rethinking strategies for drug targeting of virus-host interactions. Generalization of our approach to other RNA viruses may enable treatment of diverse infectious diseases with drug strategies that resist escape.