[Frontiers in Bioscience 8, a48-53, January 1, 2003]

Replication of respiratory syncytial virus is inhibited in target cells generating nitric oxide in situ

Dania Ali-Ahmad 1, Cynthia A. Bonville 1, Helene F. Rosenberg 2, and Joseph B. Domachowske 1

1 Department of Pediatrics, Division of Infectious Diseases, State University of New York Upstate Medical University, Syracuse, New York 13210, and 2 Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell culture
3.2. Preparation of RSV stock suspensions
3.3. Generation of HEp-2 clones that express iNOS and generate nitric oxide
3.4. Northern analysis
3.5. Nitrite assay
3.6. Plaque assays
3.7. Statistical analysis
4. Results
4.1. Characterization and iNOS expression in clonal transductants
4.2. Growth and nitrite accumulation from clonal transductants
4.3. Quantitation of nitite accumulation from exogenous NO donor
4.4. Virus replication in response to NO from endogenous vs. exogenous source
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

Nitric oxide (NO) is generated by recruited inflammatory cells and by pulmonary epithelial cells in response to respiratory virus infection, although the relative antiviral efficacy of NO from each of these sources had not been clarified. To compare the direct, antiviral potency of NO from an exogenous source with that generated by target epithelial cells in situ, we transduced HEp-2 epithelial cells with the retroviral construct, pMFGS-NOS and cloned transductant lines that generated NO constitutively. We found that NO-producing HEp-2 cells could be infected with RSV, but the titer correlated inversely with NO production, an effect that was reversed by the NOS inhibitor, NG-methyl-L-arginine (NGMMA). Our results demonstrate that NO has significant direct antiviral activity against RSV, and interestingly, that the inhibitory effect is more potent in the presence of continuous, endogenous NO production than in response to NO from an exogenous source.