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Alpha/beta interferon (IFN-␣/␤) produces antiviral effects through upregulation of many interferon-stimulated genes (ISGs) whose protein products are effectors of the antiviral state. Previous data from our laboratory have shown that IFN-␣/␤ can limit Sindbis virus (SB) replication through protein kinase R (PKR)-dependent and PKR-independent mechanisms and that one PKR-independent mechanism inhibits translation of the infecting virus genome (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). Further, using Affymetrix microarray technology, we identified 44 genes as candidates for PKR/RNase L-independent IFN-induced antiviral activities. In the current studies, we have begun analyzing these gene products for antialphavirus activity using three techniques: (i) overexpression of the protein from SB vectors and assessment of virulence attenuation in mice; (ii) overexpression of the proteins in a stable tetracycline-inducible murine fibroblast culture system and assessment of effects upon SB replication; and (iii) small interfering RNA-mediated knockdown of gene mRNA in fibroblast cultures followed by SB replication assessment as above. Tested proteins included those we hypothesized had potential to affect virus genome translation and included murine ISG20, ISG15, the zinc finger antiviral protein (ZAP), viperin, p56, p54, and p49. Interestingly, the pattern of antiviral activity for some gene products was different between in vitro and in vivo assays. Viperin and ZAP attenuated virulence most profoundly in mice. However, ISG20 and ZAP potently inhibited SB replication in vitro, whereas and viperin, p56, and ISG15 exhibited modest replication inhibition in vitro. In contrast, p54 and p49 had little to no effect in any assay.

Attachment of Sindbis virus to the cell surface glycosaminoglycan heparan sulfate (HS) and the selection of this phenotype by cell culture adaptation were investigated. Virus (TR339) was derived from a cDNA clone representing the consensus sequence of strain AR339 (K. L. McKnight, D. A. Simpson, S. C. Lin, T. A. Knott, J. M. Polo, D. F. Pence, D. B. Johannsen, H. W. Heidner, N. L. Davis, and R. E. Johnston, J. Virol. 70:1981–1989, 1996) and from mutant clones containing either one or two dominant cell culture adaptations in the E2 structural glycoprotein (Arg instead of Ser at E2 position 1 [designated TRSB]) or this mutation plus Arg for Ser at E2 114 [designated TRSB-R114]). The consensus virus, TR339, bound to baby hamster kidney (BHK) cells very poorly. The mutation in TRSB increased binding 10- to 50-fold, and the additional mutation in TRSB-R114 increased binding 3- to 5-fold over TRSB. The magnitude of binding was positively correlated with the degree of cell culture adaptation and with attenuation of these viruses in neonatal mice. HS was identified as the attachment receptor for the mutant viruses by the following experimental results. (i) Low concentrations of soluble heparin inhibited plaque formation on and binding of mutant viruses to BHK cells by >95%. In contrast, TR339 showed minimal inhibition at high concentrations. (ii) Binding and infectivity of TRSB-R114 was sensitive to digestion of cell surface HS with heparinase III, and TRSB was sensitive to both heparinase I and heparinase III. TR339 infectivity was only slightly affected by either digestion. (iii) Radiolabeled TRSB and TRSB-R114 attached efficiently to heparin-agarose beads in binding assays, while TR339 showed virtually no binding. (iv) Binding and infectivity of TRSB and TRSB-R114, but not TR339, were greatly reduced on Chinese hamster ovary cells deficient in HS specifically or all glycosaminoglycans. (v) High-multiplicity-of-infection passage of TR339 on BHK cell cultures resulted in rapid coselection of high-affinity binding to BHK cells and attachment to heparin-agarose beads. Sequencing of the passaged virus population revealed a mutation from Glu to Lys at E2 70, a mutation common to many laboratory strains of Sindbis virus. These results suggest that TR339, the most virulent virus tested, attaches to cells through a low-affinity, primarily HS-independent mechanism. Adaptive mutations, selected during cell culture growth of Sindbis virus, enhance binding and infectivity by allowing the virus to attach by an alternative mechanism that is dependent on the presence of cell surface HS.

No abstract

Yellow fever (YF) is a viral disease, endemic to tropical regions of Africa and the Americas. YF principally affects humans and nonhuman primates, and is transmitted via the bite of infected mosquitoes. The agent of YF, yellow fever virus (YFV), can cause devastating epidemics of potentially fatal, hemorrhagic disease. We rely on mass vaccination campaigns to prevent and control these outbreaks. However, the risk of major YF epidemics, especially in densely populated, poor urban settings, both in Africa and South America, has greatly increased due to: (1) reinvasion of urban settings by the mosquito vector of YF, Aedes aegypti; (2) rapid urbanization, particularly in parts of Africa, with populations shifting from rural to predominantly urban; and (3) waning immunization coverage. Consequently, YF is considered an emerging, or reemerging disease of considerable importance.