Absence Of Interferon Treatment Research Articles

Macaque interferon-induced transmembrane proteins limit replication of SHIV strains in an Envelope-dependent manner.

HIV-1 does not persistently infect macaques due in part to restriction by several macaque host factors. This has been partially circumvented by generating chimeric SIV/HIV-1 viruses (SHIVs) that encode SIV antagonist of known restriction factors. However, most SHIVs replicate poorly in macaques unless they are further adapted in culture and/or macaques (adapted SHIVs). Therefore, development of SHIVs encoding HIV-1 sequences derived directly from infected humans without adaptation (unadapted SHIVs) has been challenging. In contrast to the adapted SHIVs, the unadapted SHIVs have lower replication kinetics in macaque lymphocytes and are sensitive to type-1 interferon (IFN). The HIV-1 Envelope (Env) in the chimeric virus determines both the reduced replication and the IFN-sensitivity differences. There is limited information on macaque restriction factors that specifically limit replication of the more biologically relevant, unadapted SHIV variants. In order to identify the IFN-induced host factor(s) that could contribute to the inhibition of SHIVs in macaque lymphocytes, we measured IFN-induced gene expression in immortalized pig-tailed macaque (Ptm) lymphocytes using RNA-Seq. We found 147 genes that were significantly upregulated upon IFN treatment in Ptm lymphocytes and 31/147 were identified as genes that encode transmembrane helices and thus are likely present in membranes where interaction with viral Env is plausible. Within this group of upregulated genes with putative membrane-localized proteins, we identified several interferon-induced transmembrane protein (IFITM) genes, including several previously uncharacterized Ptm IFITM3-related genes. An evolutionary genomic analysis of these genes suggests the genes are IFITM3 duplications not found in humans that are both within the IFITM locus and also dispersed elsewhere in the Ptm genome. We observed that Ptm IFITMs are generally packaged at higher levels in unadapted SHIVs when compared to adapted SHIVs. CRISPR/Cas9-mediated knockout of Ptm IFITMs showed that depletion of IFITMs partially rescues the IFN sensitivity of unadapted SHIV. Moreover, we found that the depletion of IFITMs also increased replication of unadapted SHIV in the absence of IFN treatment, suggesting that Ptm IFITMs are likely important host factors that limit replication of unadapted SHIVs. In conclusion, this study shows that Ptm IFITMs selectively restrict replication of unadapted SHIVs. These findings suggest that restriction factors including IFITMs vary in their potency against different SHIV variants and may play a role in selecting for viruses that adapt to species-specific restriction factors.

EPSTI1 Is Involved in IL-28A-Mediated Inhibition of HCV Infection.

It has been reported that IFN-λs inhibit HCV replication in vitro. But the mechanisms of how IL-28A conducts antiviral activity and the functions of IL-28A-induced ISGs (IFN-stimulated genes) are not fully understood. In this study, we found that IL-28A has the antiviral effect on HCV life cycle including viral replication, assembly, and release. IL-28A and IFN-α synergistically inhibit virus replication. EPSTI1 (epithelial-stromal interaction 1), one of IL-28A-induced ISGs, plays a vital role in IL-28A-mediated antiviral activity. Furthermore, forced expression of EPSTI1 effectively inhibits HCV replication in the absence of interferon treatment, and knockdown of EPSTI1 contributes to viral enhancement. EPSTI1 can activate PKR promoter and induce several PKR-dependent genes, including IFN-β, IFIT1, OAS1, and RNase L, which is responsible for EPSTI1-mediated antiviral activity.

44: Interferon-dependent increase in STAT1, STAT2, and IRF9 prolongs resistance to viruses and DNA damage

A single high dose of IFN β activates powerful cellular responses in which many antiviral, pro-apoptotic, and anti-proliferative proteins are highly expressed. The expression of IFN-stimulated genes (ISGs) is mediated by ISGF3, consisting of IRF9 and tyrosine phosphorylated STATs 1 and 2. This initial response to IFN β is rapid and transient, so that the expression of most ISGs returns to basal levels within a day. However, the expression of some ISGs is prolonged for many days. We found that high levels of STAT1, STAT2, and IRF9 proteins that lack tyrosine phosphorylation are critical for the prolonged second phase response to IFN β . The expression of the STAT1, STAT2, and IRF9 proteins is increased by IFN β at late times (>24 h), and these proteins form a complex resembling ISGF3 (named unphosphorylated ISGF3, U-ISGF3), and bind to ISREs of target genes independently of tyrosine phosphorylation of STATs 1 and 2. U-ISGF3 induces only a subset of ISGs (20–30% of the total), which are mostly known to be antiviral genes and are also highly expressed in therapy-resistant cancer cells, but not cytotoxic ISGs. We observed that, in the absence of interferon treatment, high levels of U-ISGF3 significantly reduced the replication of many RNA viruses. High U-ISGF3 levels are correlated with resistance of cancer cells to DNA damage induced by radiation, etoposide, or doxorubicin. Knocking STAT1 and IRF9 down to decrease U-ISGF3 levels sensitized cancer cells to doxorubicin-induced DNA damage. Continuous exposure of cells to a low level of IFN β , often seen in cancers, leads to steady-state increases in the expression of only the U-ISGF3-dependent genes, with no sustained increases of IFN β -induced cytotoxic genes. Our study has elucidated that the U-ISGF3-dependent response to IFN β prolongs the antiviral effects, but leads to DNA damage resistance, which inhibits responses to cancer therapy.

Molecular Signatures Associated with Mx1-Mediated Resistance to Highly Pathogenic Influenza Virus Infection: Mechanisms of Survival

Understanding the role of host factors during lethal influenza virus infection is critical to deciphering the events that determine the fate of the host. One such factor is encoded by the Mx1 gene, which confers resistance to influenza virus infection. Here, we compared pathology and global gene expression profiles in lung tissue from BALB/c (Mx1(-)) and BALB · A2G-Mx1 mice (Mx1(+/+)) infected with the fully reconstructed 1918 pandemic influenza virus. Mx1(+/+) mice showed less tissue damage than Mx(-) animals, and pathology and mortality were further reduced by treating the mice with interferon prior to infection. Using global transcriptional profiling, we identified distinct molecular signatures associated with partial protection, complete protection, and the contribution of interferon to the host response. In the absence of interferon treatment, partial protection was characterized by the generation of an acute response with the upregulation of genes associated with apoptosis, reactive oxygen species, and cell migration. Complete protection was characterized by the downregulation of cytokine and chemokine genes previously associated with influenza virus pathogenesis. The contribution of interferon treatment to total protection in virus-infected Mx1(+/+) mice was characterized by the altered regulation of cell cycle genes. These genes were upregulated in Mx1(+/+) mice treated with interferon but downregulated in the absence of interferon treatment. Our results suggest that Mx1(+/+) mice generate a protective antiviral response by controlling the expression of key modulator molecules associated with influenza virus lethality.

RNA adenosine deaminase ADAR1 deficiency leads to increased activation of protein kinase PKR and reduced vesicular stomatitis virus growth following interferon treatment

Two size forms of ADAR1 adenosine deaminase are known, one constitutively expressed (p110) and the other interferon (IFN)-induced (p150). To test the role of ADAR1 in viral infection, HeLa cells with ADAR1 stably knocked down and 293 cells overexpressing ADAR1 were utilized. Overexpression of p150 ADAR1 had no significant effect on the yield of vesicular stomatitis virus. Likewise, reduction of p110 and p150 ADAR1 proteins to less than approximately 10 to 15% of parental levels (ADAR1-deficient) had no significant effect on VSV growth in the absence of IFN treatment. However, inhibition of virus growth following IFN treatment was approximately 1 log(10) further reduced compared to ADAR1-sufficient cells. The level of phosphorylated protein kinase PKR was increased in ADAR1-deficient cells compared to ADAR1-sufficient cells following IFN treatment, regardless of viral infection. These results suggest that ADAR1 suppresses activation of PKR and inhibition of VSV growth in response to IFN treatment.

Antiviral effect of the mammalian translation initiation factor 2α kinase GCN2 against RNA viruses

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

DNA Damage-binding Proteins and Heterogeneous Nuclear Ribonucleoprotein A1 Function as Constitutive KCS Element Components of the Interferon-inducible RNA-dependent Protein Kinase Promoter

Protein kinase regulated by RNA (PKR) plays important roles in many cellular processes including virus multiplication and cell growth, differentiation, and apoptosis. The promoter of the PKR gene possesses a novel 15-bp element designated KCS, positioned upstream of a consensus interferon (IFN)-stimulated response element, that is required for both basal and interferon-inducible transcription. Protein binding to the KCS element is not dependent upon IFN treatment and correlates with transcriptional activity of the PKR promoter. The identity of KCS-binding proteins (KBP) that selectively bind at the KCS element is largely unknown, except for the transcription factor Sp1. We now have purified KBP from HeLa cell nuclear extracts by ion-exchange and DNA-affinity chromatography steps and then identified four constituent proteins of the KBP complex by mass spectrometry and immunochemistry: KBP120 and KBP45 are the damaged DNA-binding protein subunits, p127 DDB1 and p48 DDB2, respectively; KBP100 is the transcription factor Sp1; and KBP35 is the heterogeneous nuclear ribonucleoprotein A1. The steady-state levels of these four KCS-binding proteins in human cells are not altered by IFN treatment. Components of the KBP complex bind selectively and constitutively to the KCS element in the absence of IFN treatment, both in vitro as measured by competition electrophoretic mobility shift assay (EMSA) and DNA pull-down assays and in vivo as measured by chromatin immunoprecipitation assays. Depletion of DDB2 by antisense strategy reduces KBP complex formation by EMSA. These results provide new insight into the biochemical identity and activity of proteins involved in PKR promoter function.

The molecular basis of MHC class II deficiency and transcriptional control of MHC class II gene expression

Protein kinase regulated by RNA (PKR) plays important roles in many cellular processes including virus multiplication and cell growth, differentiation, and apoptosis. The promoter of the PKR gene possesses a novel 15-bp element designated KCS, positioned upstream of a consensus interferon (IFN)-stimulated response element, that is required for both basal and interferon-inducible transcription. Protein binding to the KCS element is not dependent upon IFN treatment and correlates with transcriptional activity of the PKR promoter. The identity of KCS-binding proteins (KBP) that selectively bind at the KCS element is largely unknown, except for the transcription factor Sp1. We now have purified KBP from HeLa cell nuclear extracts by ion-exchange and DNA-affinity chromatography steps and then identified four constituent proteins of the KBP complex by mass spectrometry and immunochemistry: KBP120 and KBP45 are the damaged DNA-binding protein subunits, p127 DDB1 and p48 DDB2, respectively; KBP100 is the transcription factor Sp1; and KBP35 is the heterogeneous nuclear ribonucleoprotein A1. The steady-state levels of these four KCS-binding proteins in human cells are not altered by IFN treatment. Components of the KBP complex bind selectively and constitutively to the KCS element in the absence of IFN treatment, both in vitro as measured by competition electrophoretic mobility shift assay (EMSA) and DNA pull-down assays and in vivo as measured by chromatin immunoprecipitation assays. Depletion of DDB2 by antisense strategy reduces KBP complex formation by EMSA. These results provide new insight into the biochemical identity and activity of proteins involved in PKR promoter function.

Control of the ppp(a2'p)nA system in HeLa cells. Effects of interferon and virus infection.

HeLa cells have an unusually high level of ppp(A2'p)nA synthetase (n = 2 to greater than or equal to 4) even in the absence of interferon treatment. In accord with this ppp(A2'p)nA and ppp(A2'p)nA-mediated ribosomal RNA cleavage occur naturally in response to encephalomyocarditis virus infection in control as well as in interferon-treated cells. Despite this, in the absence of interferon treatment, encephalomyocarditis virus grows well in these cells. A possible explanation for this paradox is that the ppp(A2'p)nA dependent RNase is lost or inactivated at later times post-infection in control but not in interferon-treated cells. It appears, therefore, to be the prevention by interferon of the virus-mediated inhibition of the ppp(A2'p)n-dependent nuclease rather than the absolute level or induction of the ppp(A2'p)nA synthetase which is crucial for the activity of the ppp(A2'p)nA system in HeLa cells. These results provide evidence for a further level of control in the ppp(A2'p)nA system and show that limited ppp(A2'p)nA-mediated ribosomal RNA cleavage alone is not sufficient to cause an inhibition of virus growth.

Mechanism of interferon action Differential effect of interferon on the synthesis of simian virus 40 and reovirus polypeptides in monkey kidney cells

The effect of interferon (IFN) treatment on the synthesis of viral proteins was examined in BSC-1 monkey kidney cells singly and doubly infected with simian virus 40 (SV40) and reovirus. The pattern and amount of SV40 T antigen and reovirus λ, μ, and σ polypeptide synthesis were comparable in the singly and doubly infected cells in the absence of IFN treatment. As determined by Yakobson et al. ( Cell 12: 73–81, 1977) and Kingsman and Samuel ( Virology 101: 458–465, 1980) SV40-specific RNA synthesis is inhibited by treatment of cells with IFN before infection, but is not inhibited by IFN treatment after infection. IFN treatment initiated before infection inhibited the synthesis of both SV40 T antigen and reovirus polypeptides. However, SV40 T antigen synthesis was not inhibited in SV40 wt or tsA mutant-infected cells when treated with monkey kidney or human leukocyte IFN after infection. By contrast, reovirus polypeptide synthesis was markedly inhibited in BSC-1 cells treated with IFN after SV40 infection and superinfected with reovirus. SV40 infection did not rescue reovirus polypeptide synthesis from the inhibitory action of IFN, and reovirus infection did not alter the resistance of SV40 T antigen synthesis to IFN in cells IFN-treated after SV40 infection. Total cellular protein synthesis was not significantly affected by IFN treatment. These results suggest that the interferon-induced inhibitor(s) of protein synthesis discriminate between SV40 and reovirus mRNAs; the translation of SV40 mRNA, like most cellular mRNAs, is not affected under conditions where the IFN-induced inhibitor(s) significantly affect the translation of reovirus mRNA.