Identification and Characterization of Interferon-Induced Proteins That Inhibit Alphavirus Replication

Abstract

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.

Glycogen Synthase Kinase 3β (GSK3β) Modulates Antiviral Activity of Zinc-finger Antiviral Protein (ZAP)

“…*, p Ͻ 0.05. and PARN. 4 Further investigation is needed to detail the mechanisms by which phosphorylation of ZAP by GSK3␤ modulates its activity.…”

“…ZAP is not a universal antiviral factor because it does not inhibit the replication of viruses such as poliovirus and yellow fever virus (5). Type I interferon treatment or viral infection up-regulates ZAP expression (4,6,7), and down-regulation of endogenous ZAP enhances the expression of HIV-1 (2), suggesting that ZAP is an antiviral effector in vivo.…”

“…In addition to MMLV, ZAP inhibits the replication of HIV-1 (2), Ebola virus and Marburg virus (3), and certain alphaviruses such as Sindbis virus, Ross River virus, and Semliki Forest virus (4,5). ZAP is not a universal antiviral factor because it does not inhibit the replication of viruses such as poliovirus and yellow fever virus (5).…”

Alpha/Beta Interferon Inhibits Cap-Dependent Translation of Viral but Not Cellular mRNA by a PKR-Independent Mechanism

“…Alterations in the localization or phosphorylation state of the eIF4E have been implicated in the regulation of cap-dependent translation initiation (26,31). Finally, to test the association of stress-responsive genes with polysome fractions, we performed semiquantitative RT-PCR of mRNA from each fraction using primers specific to the murine ISG20 gene, which we found previously to be IFN inducible in MEFs and to possess anti-alphavirus effects (56). The results demonstrate that the ISG20 mRNA is minimally present in untreated cells but induced by IFN treatment and is associated with heavy polysome fractions (e.g., fractions 8 to 11), suggesting productive translation (Fig.…”

“…The IFN-inducible 2Ј-5Ј OAS binds dsRNA and synthesizes 2Ј-5Ј-linked oligoadenylates that activate constitutive RNase L to cleave host and viral single-stranded RNA. More recently, antiviral effects have also been attributed to the IFN-␣/␤-inducible proteins Mx, a tryptophan-degrading enzyme (38), adenosine deaminase (34), ISG20 (13,56), p56 (51,56), ISG15 (28,29,40), viperin (56), mGBP2 (6), GBP-1 (1), the APOBEC proteins (50), and other, as-yet-undescribed, factors (43,57).…”