Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human leukocyte interferon. I. Effect on early and late stages of the viral multiplication cycle

The kinetics of induction in human amnion U cells of the antiviral activity against vesicular stomatitis virus (VSV) produced by a single molecularly cloned subspecies of human leukocyte interferon (IFN-alpha A) were examined. IFN-alpha A-induced inhibition was found to be biphasic over a period of 24 h with the major extent of VSV inhibition occurring within the first 6 h of IFN treatment. The relationship of this major phase of inhibition to the early and late events of the VSV multiplication cycle was investigated. IFN-alpha A treatment had no detectable effect on the adsorption and penetration of VSV virions or on their uncoating to yield viral nucleocapsids. The polypeptides of adsorbed or uncoated VSV particles were neither preferentially degraded nor detectably altered in IFN-treated cells, as compared to untreated cells. Progeny virions released from IFN-treated cells, although greatly reduced in number, were found to be equally as infectious as those released from untreated cells. Progeny virions from IFN-treated cells also had a normal complement of VSV proteins in the same ratios as were seen in virions from untreated cells; specifically, IFN treatment produced no reduction in the incorporation of G or M protein into assembled virions. These results suggest that conditions of IFN treatment sufficient to reduce the yield of infectious VSV progeny greater than 99% do not detectably affect either the early or the late stages of the VSV multiplication cycle.     

Mechanism of interferon action. I. Characterization of a 54-kDa protein induced by gamma interferon with properties similar to a cytoskeletal component

A polypeptide of 54-kDa molecular mass (p54) induced by molecularly cloned human interferon gamma (rIFN-gamma) in human amnion U cells was characterized biochemically and immunologically. Rabbit polyclonal antibody to p54 was prepared using p54 purified from human amnion U cells as the immunogen. As measured by immunoprecipitation of [35S]methionine pulse-labeled extracts or by immunoblot analysis of unlabeled extracts, the synthesis of p54 was greatly elevated in three human cell lines treated with IFN-gamma, amnion U, fibroblast GM2767, and fibroblast F153. Moreover, the rabbit anti-p54 polyclonal antibody preparation cross-reacted with cytoskeleton-associated polypeptides prepared from human cells and from sea urchin embryos. By use of a monoclonal antibody probe directed against the common domain of intermediate filaments (anti-intermediate filament antibody), it was established that intermediate filament components are also induced by rIFN-gamma. Anti-p54 polyclonal antibody cross-reacted with anti-intermediate filament antibody-recognized, rIFN-gamma-induced polypeptides. Thus, it appears that p54 may be an IFN-induced cytoskeleton-associated polypeptide.     

Inhibition of L-deleted foot-and-mouth disease virus replication by alpha/beta interferon involves double-stranded RNA-dependent protein kinase

We previously demonstrated that the ability of foot-and-mouth disease virus (FMDV) to form plaques in cell culture is associated with the suppression of alpha/beta interferon (IFN-alpha/beta). In the present study, we used Escherichia coli-expressed porcine and bovine IFN-alpha or -beta individually to demonstrate that each was equally effective in inhibiting FMDV replication. The block in FMDV replication appeared to be at the level of protein translation, suggesting a role for double-stranded RNA-dependent protein kinase (PKR). In support of these findings, treatment of porcine and bovine cells with 2-aminopurine, an inhibitor of PKR, increased the yield of virus 8.8- and 11.2-fold, respectively, compared to that in untreated infected cells. In addition, results of FMDV infection in mouse embryonic fibroblast cells derived from gene knockout mice lacking the gene for RNase L(-/-) or PKR(-/-) or both indicated an important role for PKR in the inhibition of FMDV replication.     

Prophylactic alpha interferon treatment increases the therapeutic index of oncolytic vesicular stomatitis virus virotherapy for advanced hepatocellular carcinoma in immune-competent rats

Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to substantially attenuated antiviral responses in many tumors. We have recently reported that recombinant VSV vector can be used as an effective oncolytic agent to safely treat multifocal hepatocellular carcinoma (HCC) in the livers of immune-competent rats via hepatic artery infusion. When administered at doses above the maximum tolerated dose (MTD), however, the animals suffered from neurotoxicity and/or acute lethal hepatotoxicity. Since VSV is extremely sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta) in normal cells, we tested if prophylactic treatment with rat IFN-alpha would enhance VSV safety without compromising treatment efficacy in tumor-bearing rats. We found that VSV retained its replication potential in human and rat HCC cells after preincubation with relatively high doses of rat and human IFN-alpha in vitro, and its MTD in tumor-bearing rats treated systemically with rat IFN-alpha at 66 IU/g body weight (BW), equivalent to a human IFN-alpha dose that is currently prescribed for patients with viral hepatitis, was elevated by more than 1/2 log unit. Furthermore, we demonstrate that intratumoral replication of VSV was not attenuated by administration of 66 IU/g BW rat IFN-alpha, as tumor response and survival advantage in VSV-treated rats in the presence or absence of rat IFN-alpha were equivalent. The results suggest that prophylactic rat IFN-alpha treatment elevates the therapeutic index of hepatic arterial VSV therapy for multifocal HCC in rats. Since human IFN-alpha is currently in clinical use, its prophylactic application should be considered in future clinical translational protocols for VSV-mediated oncolytic virotherapy as a novel therapeutic modality in patients with advanced HCC, as well as other types of cancer.     

Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human gamma-interferon. I. Effect on early and late stages of the viral multiplication cycle

The molecular basis of the inhibition of vesicular stomatitis virus (VSV) replication by pure recombinant gamma-interferon (IFN-gamma) in human amnion U cells was examined. A saturating concentration of IFN-gamma induced, at maximum, about a two log10 reduction in infectious VSV yield. The kinetics of induction of the antiviral activity by IFN-gamma were first order over the period of about 6-18 h, following a lag of about 3 h, after treatment with a saturating concentration of IFN-gamma. The relationship of the inhibition in VSV infectivity to the early and late events of the VSV multiplication cycle was investigated. IFN-gamma treatment had no detectable effect on the adsorption and penetration of VSV virions or on their uncoating to yield viral nucleocapsids. The polypeptides of adsorbed or uncoated VSV particles were neither preferentially degraded nor detectably altered in IFN-gamma-treated U cells, as compared to untreated U cells. Progeny virions isolated from IFN-gamma-treated U cells, although greatly reduced in number, were found to be equally as infectious as those isolated from untreated U cells. Progeny virions from IFN-gamma-treated cells also possessed the same composition of viral proteins as was observed for virions from untreated cells. These results suggest that conditions of IFN-gamma treatment sufficient to reduce the yield of infectious VSV progeny 100-fold do not detectably affect either the early or the late stages of the VSV multiplication cycle.     

Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.     

Identification and characterization of interferon-induced proteins that inhibit alphavirus replication

Alpha/beta interferon (IFN-alpha/beta) 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-alpha/beta 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.     

Ebola virus VP24 binds karyopherin alpha1 and blocks STAT1 nuclear accumulation

Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-alpha/beta) and the ability of cells to respond to IFN-alpha/beta or IFN-gamma. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-alpha/beta production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-alpha/beta and IFN-gamma signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-alpha/beta and IFN-gamma signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with karyopherin alpha1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin alpha2, alpha3, or alpha4. Overexpression of VP24 results in a loss of karyopherin alpha1-PY-STAT1 interaction, indicating that the VP24-karyopherin alpha1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.     

Variable deficiencies in the interferon response enhance susceptibility to vesicular stomatitis virus oncolytic actions in glioblastoma cells but not in normal human glial cells

With little improvement in the poor prognosis for humans with high-grade glioma brain tumors, alternative therapeutic strategies are needed. As such, selective replication-competent oncolytic viruses may be useful as a potential treatment modality. Here we test the hypothesis that defects in the interferon (IFN) pathway could be exploited to enhance the selective oncolytic profile of vesicular stomatitis virus (VSV) in glioblastoma cells. Two green fluorescent protein-expressing VSV strains, recombinant VSV and the glioma-adapted recombinant VSV-rp30a, were used to study infection of a variety of human glioblastoma cell lines compared to a panel of control cells, including normal human astrocytes, oligodendrocyte precursor cells, and primary explant cultures from human brain tissue. Infection rate, cell viability, viral replication, and IFN-alpha/beta-related gene expression were compared in the absence and presence of IFN-alpha or polyriboinosinic polyribocytidylic acid [poly(I:C)], a synthetic inducer of the IFN-alpha/beta pathway. Both VSV strains caused rapid and total infection and death of all tumor cell lines tested. To a lesser degree, normal cells were also subject to VSV infection. In contrast, IFN-alpha or poly(I:C) completely attenuated the infection of all primary control brain cells, whereas most glioblastoma cell lines treated with IFN-alpha or poly(I:C) showed little or no sign of protection and were killed by VSV. Together, our results demonstrate that activation of the interferon pathway protects normal human brain cells from VSV infection while maintaining the vulnerability of human glioblastoma cells to viral destruction.     

Synergistic antiviral and antiproliferative activities of Escherichia coli-derived human alpha, beta, and gamma interferons

The antiviral and antiproliferative effects of highly purified Escherichia coli-derived human interferons (IFNs) were examined in human melanoma cells (Hs294T). Antiproliferative activity was monitored by measuring inhibition of cell multiplication, and antiviral activity was determined by inhibition of herpes simplex virus type 1 replication. Treatment of cells with IFN-gamma in combination with IFN-alpha A or IFN-beta 1 resulted in potentiation of both antiproliferative and antiviral activities. In contrast, combination treatments composed of IFN-alpha A and IFN beta 1 yielded inconsistent results. Some combinations reflected additive responses, whereas others were antagonistic. To examine correlations between IFN-induced biological activities and interactions of the different IFNs with cell surface receptors, in vivo [35S]methionine-labeled IFN-alpha A was prepared. Binding studies indicated the presence of 2,980 +/- 170 receptors per cell, each with an apparent Kd of (8.4 +/- 1.3) X 10(-11) M. Results from competitive binding studies suggested that Hs294T cells possess at least two types of IFN receptors: one which binds IFN-alpha A and IFN-beta 1 and another to which IFN-gamma binds.     

Alpha interferon inhibits early stages of the human immunodeficiency virus type 1 replication cycle.

In this study, we have analyzed the effect of human alpha interferon (IFN-alpha) on a single replication cycle of human immunodeficiency virus type 1 (HIV-1) infection in the lymphocytic cell line CEM-174, which is highly sensitive to the antiviral effects of IFN. Pretreatment of cells with 50 to 500 U of recombinant human IFN-alpha per ml resulted in a marked reduction in viral RNA and protein synthesis. The effect of IFN-alpha was dose dependent and was amplified in multiple infection cycles. IFN-induced inhibition of viral protein synthesis could be detected only when cells were treated with IFN-alpha prior to infection or when IFN-alpha was added up to 10 h postinfection, but not if IFN-alpha was added at the later stages of HIV-1 replication cycle or after the HIV-1 infection was already established. Analysis of the integrated HIV-1 provirus showed a marked decrease in the levels of proviral DNA in IFN-treated cells. Thus, in contrast to the previous studies on established HIV-1 infection in T cells, in which the IFN block appeared to be at the posttranslational level, during de novo infection, IFN-alpha interferes with an early step of HIV-1 replication cycle that occurs prior to the integration of the proviral DNA. These results indicate that the early IFN block of HIV-1 replication, which has been previously observed only in primary marcophages, can also be detected in the IFN-sensitive T cells, indicating that the early IFN block is not limited to macrophages.     

Sindbis virus translation is inhibited by a PKR/RNase L-independent effector induced by alpha/beta interferon priming of dendritic cells

The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-alpha/beta)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol. 15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-alpha/beta treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-alpha/beta priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1-/-, and TD mice following infection with SB or treatment with IFN-alpha/beta. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-alpha/beta treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-alpha/beta-treated IFNAR1-/- cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.