A recombinant vaccinia virus encoding inducible nitric oxide synthase is attenuated in vivo

Abstract

To investigate the role of nitric oxide during vaccinia virus (VV) infection of mice, a recombinant VV encoding the inducible nitric oxide synthase (iNOS) gene (VV-HA-iNOS) was constructed. Following infection of immunocompromised or immunocompetent mice, the virus was highly attenuated compared with a control recombinant VV. Athymic and sublethally irradiated mice survived infection with 10 7 PFU of VV-HA-iNOS, a dose that resulted in uniform mortality in mice infected with the control recombinant VV. Attenuated virus growth was evident as early as 24 h following infection, suggesting that NO had direct antiviral activity. We have previously shown that treatment of mice with the inhibitor of NO production N G-methyl-L-arginine did not influence the course of VV infection in mice. The present study has indicated that NO can potentially exert an antiviral effect during murine VV infection. We propose that during VV infection, nitric oxide production contributes to the control of virus growth, but that in its absence, other antiviral mechanisms are sufficient to mediate fully effective virus clearance. MATERIALS AND METHODS Mice. CBA/H and Swiss athymic nude mice were obtained from the Animal Breeding Establishment of the John Curtin School of Medical Research. The mice were bred under specific-pathogen-free conditions and were used at 6 to 8 weeks of age. Materials. The human osteosarcoma cell line 143B (48), the monkey kidney cell line CV-1 (24), the murine fibroblast line L929 (50), and the Moloney leukemia virus-induced murine lymphoma line YAC-1 (29) were grown in Eagle's minimum essential medium (GIBCO, Grand Island, N.Y.) supplemented with 5% fetal calf serum, 10 mM HEPES (N-2-hydroxyethylpiperazine-NЈ-2ethanesulfonic acid), 1 mM glutamine, and antibiotics. N G-methyl-L-arginine (NMA) was synthesized as previously described (46). Purity was assessed by thin-layer chromatography and microanalysis (percentages of C, H, and N within 0.2% of required values). NMA dose-dependently inhibited NO production (see Fig. 4B). Construction of VV-HA-iNOS. Murine macrophage iNOS cDNA in plasmid CL-BS-mac-NOS (38) was provided by Charles Lowenstein (Johns Hopkins University, Baltimore, Md.). A 4,100-bp fragment containing the iNOS cDNA was excised from CL-BS-mac-NOS by a NotI digest, and this fragment was subjected to a further HincII digest to remove 136 bp from the 5Ј end. The protruding 5Ј ends of the DNA were filled by using the Klenow fragment of Escherichia coli DNA polymerase I, and the iNOS cDNA was then cloned into the HincII-digested vector pPS7.5A (14) under the control of the early-late P7.5 VV promoter. Restriction analysis with BamHI and ClaI was used to confirm correct orientation of the iNOS gene. The resulting plasmid was designated pPS7.5A-iNOS (Fig. 1). An rVV expressing iNOS was constructed from the thymidine kinase (TK)negative virus VV-HA-PR8 (13) by using marker rescue of the TK gene (8) and methotrexate selection (45). The presence of the iNOS gene was determined by dot blot hybridization with 32 P-l...

Importance of the cysteine-rich carboxyl-terminal half of V protein for Sendai virus pathogenesis

“…In this context, SeV V protein is reminiscent of several nonessential gene products encoded by large DNA viruses such as herpes and vaccinia viruses (14,33). Early defense may also be caused by induction of NO to kill and eliminate infected cells (26,34). The attack by NO is associated with elevation of intracellular levels of divalent cations (10,21), and the cations need to be chelated for the cells to survive.…”

Cytokines and immunity to viral infections

“…The activity of NO during W infection was investigated furdier using a recombinant VV encoding iNOS (W-iNOS). Tissue culture cells infected with VV-iNOS produced high levels of NO, resulting in a considerable reduction in levels of virus replication, W-iNOS was also liighly attenuated in immunodeficient mice, clearly demonstrating, in vivo, the antiviral activity of NO (96).…”

Identification ofNitric Oxide Synthase 2as an Innate Resistance Locus against Ectromelia Virus Infection

“…In those studies, transfection of the NOS2 gene restricted VV replication in vitro (11,20) and attenuated replication of NOS2 encoding recombinant VV in vivo (20). Yet administration of L-NMA failed to render B6, CBA/H, or nude mice more susceptible (20,21). In the latter mouse strains, other IFN-␥dependent pathways may have compensated for the loss of NO, as suggested by experiments in which healthy NOS2deficient animals infected with VV become vulnerable once they are treated with anti-IFN-␥ antibodies (11a).…”