Differential Inhibition of HIV Replication by the 12 Interferon Alpha Subtypes.

Type I interferons (IFNs) are a family of cytokines that represent a first line of defense against virus infections. The 12 different IFN-α subtypes share a receptor on target cells and trigger similar signaling cascades. Several studies have collectively shown that this apparent redundancy conceals qualitatively different responses induced by individual subtypes, which display different efficacies of inhibition of HIV replication. Some studies, however, provided evidence that the disparities are quantitative rather than qualitative. Since RNA expression analyses show a large but incomplete overlap of the genes induced, they may support both models. To explore if the IFN-α subtypes induce functionally relevant different anti-HIV activities, we have compared the efficacies of inhibition of all 12 subtypes on HIV spread and on specific steps of the viral replication cycle, including viral entry, reverse transcription, protein synthesis, and virus release. Finding different hierarchies of inhibition would validate the induction of qualitatively different responses. We found that while most subtypes similarly inhibit virus entry, they display distinctive potencies on other early steps of HIV replication. In addition, only some subtypes were able to target effectively the late steps. The extent of induction of known anti-HIV factors helps to explain some, but not all differences observed, confirming the participation of additional IFN-induced anti-HIV effectors. Our findings support the notion that different IFN-α subtypes can induce the expression of qualitatively different antiviral activities. IMPORTANCE The initial response against viruses relies in large part on type I interferons, which include 12 subtypes of IFN-α. These cytokines bind to a common receptor on the cell surface and trigger the expression of incompletely overlapping sets of genes. Whether the anti-HIV responses induced by IFN-α subtypes differ in the extent of expression or in the nature of the genes involved remains debated. Also, RNA expression profiles led to opposite conclusions, depending on the importance attributed to the induction of common or distinctive genes. To explore if relevant anti-HIV activities can be differently induced by the IFN-α subtypes, we compared their relative efficacies on specific steps of the replication cycle. We show that the hierarchy of IFN potencies depends on the step analyzed, supporting qualitatively different responses. This work will also prompt the search for novel IFN-induced anti-HIV factors acting on specific steps of the replication cycle.

Qualitative Differences Between the IFNα subtypes and IFNß Influence Chronic Mucosal HIV-1 Pathogenesis.

The Type I Interferons (IFN-Is) are innate antiviral cytokines that include 12 different IFNα subtypes and IFNß that signal through the IFN-I receptor (IFNAR), inducing hundreds of IFN-stimulated genes (ISGs) that comprise the 'interferome'. Quantitative differences in IFNAR binding correlate with antiviral activity, but whether IFN-Is exhibit qualitative differences remains controversial. Moreover, the IFN-I response is protective during acute HIV-1 infection, but likely pathogenic during the chronic stages. To gain a deeper understanding of the IFN-I response, we compared the interferomes of IFNα subtypes dominantly-expressed in HIV-1-exposed plasmacytoid dendritic cells (1, 2, 5, 8 and 14) and IFNß in the earliest cellular targets of HIV-1 infection. Primary gut CD4 T cells from 3 donors were treated for 18 hours ex vivo with individual IFN-Is normalized for IFNAR signaling strength. Of 1,969 IFN-regulated genes, 246 'core ISGs' were induced by all IFN-Is tested. However, many IFN-regulated genes were not shared between the IFNα subtypes despite similar induction of canonical antiviral ISGs such as ISG15, RSAD2 and MX1, formally demonstrating qualitative differences between the IFNα subtypes. Notably, IFNß induced a broader interferome than the individual IFNα subtypes. Since IFNß, and not IFNα, is upregulated during chronic HIV-1 infection in the gut, we compared core ISGs and IFNß-specific ISGs from colon pinch biopsies of HIV-1-uninfected (n = 13) versus age- and gender-matched, antiretroviral-therapy naïve persons with HIV-1 (PWH; n = 19). Core ISGs linked to inflammation, T cell activation and immune exhaustion were elevated in PWH, positively correlated with plasma lipopolysaccharide (LPS) levels and gut IFNß levels, and negatively correlated with gut CD4 T cell frequencies. In sharp contrast, IFNß-specific ISGs linked to protein translation and anti-inflammatory responses were significantly downregulated in PWH, negatively correlated with gut IFNß and LPS, and positively correlated with plasma IL6 and gut CD4 T cell frequencies. Our findings reveal qualitative differences in interferome induction by diverse IFN-Is and suggest potential mechanisms for how IFNß may drive HIV-1 pathogenesis in the gut.

Diversity of locally produced IFN-α subtypes in human nasopharyngeal epithelial cells and mouse lung tissues during influenza virus infection.

The excessively expressed interferon-α (IFN-α) might contribute to the uncontrolled inflammatory responses, causing pathological damage during influenza virus infection. However, the correlation of the pathological damage with the expression profile of IFN-α subtypes in the focus of infection with influenza viruses is poorly understood. To investigate this, we detected the IFN-α subtype dominance in human respiratory epithelial cells and mouse lungs, both of which were infected with influenza viruses. It was found that IFN-α1, IFN-α6, IFN-α14, and IFN-α16 were dominantly expressed in respiratory epithelial cells from the patients infected with IAV, whereas IFN-α5, IFN-α8, and IFN-α21 were dominantly expressed in respiratory epithelial cells from the patients infected with less pathogenic IBV and that IFN-α1, IFN-α9, and IFN-α15 were dominantly expressed in lungs of the mice infected with H1N1 IAV, and IFN-α2, IFN-α12, and IFN-α13 were dominantly expressed in lungs of the mice infected with less pathogenic H9N2 IAV. Compared with H9N2 IAV, H1N1 IAV induced higher mortality rates and more obvious body weight loss in the mice. In addition, IAV or H1N1 IAV induced a significantly higher level of CXCL10 mRNA in the human respiratory epithelial cells or the mouse lungs, respectively. In mice, the high level of Cxcl10 mRNA was accompanied by the abundant infiltrated neutrophils and more severe pathological changes in the lungs. Together, the data presented here indicate that the pathogenicity of influenza viruses is correlated with the IFN-α subtypes induced by influenza viruses. KEY POINTS: • Different influenza viruses induce differential inflammation responses. • Various influenza viruses induce diverse expression profiles of IFN-α subtypes. • The locally produced IFN-α subtypes correlated to the differential inflammation. Graphical abstract.

Interferon Alpha Subtype-Specific Suppression of HIV-1 Infection In Vivo.

UNLABELLED: Although all 12 subtypes of human interferon alpha (IFN-α) bind the same receptor, recent results have demonstrated that they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFN-α2 subtype is currently in HIV-1 clinical trials, but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1 in vitro We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFN-α14 subtype has very potent anti-HIV-1 activity whereas IFN-α2 does not. In both postexposure prophylaxis and treatment of acute infections, IFN-α14, but not IFN-α2, significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyperactivation, which is a prognosticator of disease progression, was reduced by IFN-α14 but not IFN-α2. Whereas ineffective IFN-α2 therapy was associated with CD8(+) T cell activation, successful IFN-α14 therapy was associated with increased intrinsic and innate immunity, including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL(+) NK cells. These results identify IFN-α14 as a potent new therapeutic that operates via mechanisms distinct from those of antiretroviral drugs. The ability of IFN-α14 to reduce both viremia and proviral loads in vivo suggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFN-α subtype should be evaluated against the specific virus being treated. IMPORTANCE: The naturally occurring antiviral protein IFN-α2 is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFN-α acts by different mechanism than ARVs and has been shown to reduce HIV proviral loads, clinical trials are under way to test whether IFN-α2 combined with ARVs might eradicate HIV-1 infections. IFN-α is actually a family of 12 distinct proteins, and each IFN-α subtype has different efficacies toward different viruses. Here, we use mice that contain a human immune system, so they can be infected with HIV. With this model, we demonstrate that while IFN-α2 is only weakly effective against HIV, IFN-α14 is extremely potent. This discovery identifies IFN-α14 as a more powerful IFN-α subtype for use in combination therapy trials aimed toward an HIV cure.

Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes.

UNLABELLED: Type I interferons (IFNs) are induced upon viral infection and important mediators of innate immunity. While there is 1 beta interferon (IFN-ß) protein, there are 12 different IFN-α subtypes. It has been reported extensively that different viruses induce distinct patterns of IFN subtypes, but it has not been previously shown how the viral multiplicity of infection (MOI) can affect IFN induction. In this study, we discovered the novel finding that human U937 cells infected with 2 different concentrations of Sendai virus (SeV) induce 2 distinct type I IFN subtype profiles. Cells infected at the lower MOI induced more subtypes than cells infected at the higher MOI. We found that this was due to the extent of signaling through the IFN receptor (IFNAR). The cells infected at the lower viral MOI induced the IFNAR2-dependent IFN-α subtypes 4, 6, 7, 10, and 17, which were not induced in cells infected at higher virus concentrations. IFN-ß and IFN-α1, -2, and -8 were induced in an IFNAR-independent manner in cells infected at both virus concentrations. IFN-α5, -14, -16, and -21 were induced in an IFNAR-dependent manner in cells infected at lower virus concentrations and in an IFNAR-independent manner in cells infected at higher virus concentrations. These differences in IFN subtype profiles in the 2 virus concentrations also resulted in distinct interferon-stimulated gene induction. These results present the novel finding that different viral MOIs differentially activate JAK/STAT signaling through the IFNAR, which greatly affects the profile of IFN subtypes that are induced. IMPORTANCE: Type I IFNs are pleiotropic cytokines that are instrumental in combating viral diseases. Understanding how the individual subtypes are induced is important in developing strategies to block viral replication. Many studies have reported that different viruses induce distinct type I IFN subtype profiles due to differences in the way viruses are sensed in different cell types. However, we report in our study the novel finding that the amount of virus used to infect a system can also affect which type I IFN subtypes are induced due to the extent of activation of certain signaling pathways. These distinct IFN subtype profiles in cells infected at different MOIs are correlated with differences in interferon-stimulated gene induction, indicating that the same virus can induce distinct antiviral responses depending on the MOI. Because type I IFNs are used as therapeutic agents to treat viral diseases, understanding their antiviral mechanisms can enhance clinical treatments.

Binding and activity of all human alpha interferon subtypes.

Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5-5 µM and 0.4-5 nM respectively (except for IFN-alpha1 - 220 nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC50 values differ significantly (1.5 nM versus 0.1 nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system.

The differential activity of interferon-α subtypes is consistent among distinct target genes and cell types.

IFN-α proteins have been described to originate from 14 individual genes and allelic variants. However, the exceptional diversity of IFN-α and its functional impact are still poorly understood. To characterize the biological activity of IFN-α subtypes in relation to the cellular background, we investigated the effect of IFN-α treatment in primary fibroblasts and endothelial cells of vascular or lymphatic origin. The cellular response was evaluated for 13 distinct IFN-α proteins with respect to transcript regulation of the IFN-stimulated genes (ISGs) IFIT1, ISG15, CXCL10, CXCL11 and CCL8. The IFN-α proteins displayed a remarkably consistent potency in gene induction irrespective of target gene and cellular background which led to the classification of IFN-α subtypes with low (IFN-α1), intermediate (IFN-α2a, -4a, -4b, -5, -16, -21) and high (IFN-α2b, -6, -7, -8, -10, -14) activity. The differential potency of IFN-α classes was confirmed at the ISG protein level and the functional protection of cells against influenza virus infection. Differences in IFN activity were only observed at subsaturating levels of IFN-α proteins and did not affect the time course of ISG regulation. Cell-type specific responses were apparent for distinct target genes independent of IFN-α subtype and were based on different levels of basal versus inducible gene expression. While fibroblasts presented with a high constitutive level of IFIT1, the expression in endothelial cells was strongly induced by IFN-α. In contrast, CXCL10 and CXCL11 transcript levels were generally higher in endothelial cells despite a pronounced induction by IFN-α in fibroblasts. In summary, the divergent potency of IFN-α proteins and the cell-type specific regulation of individual IFN target genes may allow for the fine tuning of cellular responses to pathogen defense.

Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities.

Influenza is an acute respiratory infection causing high morbidity and mortality in annual outbreaks worldwide. Antiviral drugs are limited and pose the risk of resistance development, calling for new treatment options. IFN-α subtypes are immune-stimulatory cytokines with strong antiviral activities against IAV in vitro and in vivo. However, the clinical use of IFN-α2, the only licensed subtype of this multi-gene family, could not prevent or limit IAV infections in humans. However, the other subtypes were not investigated.Therefore, this study evaluated the induction and antiviral potential of all human IFN-α subtypes during H3N2 IAV infection in human lung explants. We found that subtypes with weak antiviral activities were preferentially induced during IAV infection in human lungs. Intriguingly, non-induced subtypes α16, α5 and α4 suppressed viral replication up to 230-fold more efficiently than α2. Furthermore, our results demonstrate that subtypes with stronger antiviral activities induce higher expression of IAV-specific restriction factors and that MxA expression is a determinant of the subtype-specific antiviral activity towards H3N2 IAV. These results corroborate that IFN-α subtypes exhibit differential antiviral activities and emphasize that subtypes α16, α5 and α4 should be further investigated for the prevention and treatment of severe infections with seasonal H3N2 IAV.

Diverse Immunomodulatory Effects of Individual IFNα Subtypes on Virus-Specific CD8+ T Cell Responses.

Clinical administration of Interferon α (IFNα) resulted in limited therapeutic success against some viral infections. Immune modulation of CD8+ T cell responses during IFNα therapy is believed to play a pivotal role in promoting viral clearance. However, these clinical studies primarily focused on IFNα subtype 2. To date, the immunomodulatory roles of the remaining 10-13 IFNα subtypes remains poorly understood, thereby precluding assessments of their potential for more effective treatments. Here, we report that virus-specific CD8+ T cell responses were influenced to various extents by individual IFNα subtypes. IFNα4, 6, and 9 had the strongest effects on CD8+ T cells, including antiproliferative effects, improved cytokine production and cytotoxicity. Interestingly, augmented cytokine responses were dependent on IFNα subtype stimulation of dendritic cells (DCs), while antiproliferative effects and cytotoxicity were mediated by IFNAR signaling in either CD8+ T cells or DCs. Thus, precise modulation of virus-specific CD8+ T cell responses may be feasible for specific antiviral immunotherapies through careful selection and administration of individual IFNα subtypes.

Dose-Dependent Differences in HIV Inhibition by Different Interferon Alpha Subtypes While Having Overall Similar Biologic Effects.

Type I interferons (IFNs) are key players in the antiviral immune response. Interferon alpha (IFN-α) belongs to this class of IFNs and comprises 12 subtypes that differ from each other in their binding affinities for a common receptor and, thus, in their signaling potencies. Recent data suggest that IFN-α6 and -α14 are the most potent IFN-α subtypes in restricting HIV replication when applied exogenously. However, in the context of antiviral therapy, IFNs are administered at high doses, which may compensate for differences in potency seen between IFN-α subtypes. In this study, we reexamined whether IFN-α subtypes induce different biological activities, with a focus on how IFN-α treatment dose affects cellular responses to HIV in primary CD4+ T cells, peripheral blood mononuclear cells (PBMCs), and macrophages. We found that the subtypes' antiviral activities were dose dependent, with >90% inhibition of HIV replication at a high dose of all IFN-αs except the weak IFN-α/ß receptor (IFNAR) binder, IFN-α1. The quality of the responses engendered by IFN-α1, -α2, -α6, and -α14 was highly comparable, with essentially the same set of genes induced by all four subtypes. Hierarchal cluster analysis revealed that the individual donors were stronger determinants for the IFN-stimulated-gene (ISG) responses than the specific IFN-α subtype used for stimulation. Notably, IFN-α2-derived mutants with substantially reduced IFNAR2 binding still inhibited HIV replication efficiently, whereas mutants with increased IFNAR1 binding potentiated antiviral activity. Overall, our results support the idea that IFN-α subtypes do not induce different biological responses, given that each subtype is exogenously applied at bioequivalent doses.IMPORTANCE Elucidating the functional role of the IFN-α subtypes is of particular importance for the development of efficacious therapies using exogenous IFN-α. Specifically, this will help define whether IFN therapy should be based on the use of pathogen-dependent IFN subtypes or, rather, IFN mutants with optimized IFNAR binding properties.

Detection of differential expression of porcine IFN-alpha subtypes by reverse transcription polymerase chain reaction.

The porcine interferon-alpha (IFN-alpha) multigene family is a new IFN-alpha system in recent research. Characterization of the PoIFN-alpha multigene family has been described in our previous work, and 14 functional PoIFN-alpha genes were detected in the porcine genome. In this study, we designed subtype-specific primers and consensus primers for PoIFN-alpha. The expression of PoIFN-alpha was detected using the two PCR strategies in three systems, namely, poly(I).poly(C)-DEAE-dextran-induced PK15 cells, pseudorabies virus-infected PK15 cells, and infected PK15 cells with an attenuated strain of swine fever virus, respectively. In poly(I).poly(C)-DEAE-dextran-induced PK15 cells, the expression of IFN-alpha2, -alpha3, -alpha4, -alpha8, and -alpha9 after 6-h/24-h inducement in PK15 cells were observed. In pseudorabies virus-infected PK15 cells, the expression of PoIFN-alpha2, -alpha3, -alpha8, -alpha9, -alpha10, and -alpha13 was observed after 6-h/24-h infection, and in the attenuated strain of swine fever virus-infected PK15 cells, upregulation of PoIFN-alpha2, -alpha3, -alpha4, -alpha8, -alpha9, and -alpha10 was detected. The results of realtime quantitative PCR analysis suggested that the expression was time-dependent in pseudorabies virus/poly(I).poly(C)-DEAE-dextran-induced PK15 cells, but in the attenuated swine fever virus-infected PK15 system, the expression level of IFN-alpha subtypes was not obviously time dependent.

The combination of IFN-alpha2 and IFN-alpha8 exhibits synergistic antiproliferative activity on renal cell carcinoma (RCC) cell lines through increased binding affinity for IFNAR-2.

Although there are at least 13 interferon-alpha (IFN-alpha) subtypes in humans, interactions between the subtypes remain unknown. To understand IFN-alpha interactions, we examined the antiproliferative activities and the receptor binding affinities of different combinations of IFN-alpha2 and IFN-alpha8 using six renal cell carcinoma (RCC) cell lines. Although IFN-alpha8 was the more potent subtype, synergistic and antagonistic antiproliferative effects were also observed in certain combinations of IFN-alpha2 and IFN-alpha8. To analyze the interactions between IFN-alpha2 and IFN-alpha8, the receptor-binding kinetics of different combinations of IFN-alpha2 and IFN- alpha8 to the IFN-alpha receptors, IFNAR-1 or IFNAR-2, were measured using a surface plasmon resonance-based biosensor. Unexpectedly, the receptor binding kinetics to IFNAR-2 but not to IFNAR-1 were mutually related to antiproliferative activity and increase in the binding speed (K(a)) for IFNAR-2. Moreover, we observed the increased fluorescence intensity (FI) of biotin-labeled IFN-alpha8 to IFNAR-2 by receptor binding inhibition assay with unlabeled IFN-alpha2 but not the other combinations. These findings indicate that the binding manner of IFN-alpha8 for IFNAR-2 is different from that of IFN-alpha2, suggesting that binding of IFN-alpha8 rather than binding of IFN-alpha2 to IFNAR-2 leads to activation and subsequent antiproliferative activity despite the same antiviral activity in RCC.

Expression Pattern of Individual IFNA Subtypes in Chronic HIV Infection.

Interferon-α (IFN-α) plays an important role in HIV pathogenesis. IFN-α consists of 13 individual IFN-α subtypes, which exhibit individual antiviral and immunomodulatory activities in HIV infection. Here, we determined the expression profiles of all IFN-α subtypes in treated and treatment-naive HIV+ patients and their impact on the induction of distinct HIV restriction factors. We collected blood samples of chronic HIV+ patients, which underwent antiretroviral therapy or were treatment-naive, and determined the individual expression levels of different IFN-α subtypes and HIV restriction factors. HIV infection transiently enhanced the expression of IFNA mRNA. The IFN-α response was dominated by the most abundantly expressed subtypes IFNA4, A5, A7, and A14 in all individuals. HIV infection affected the expression pattern of the IFN-α response, in particular for IFNA2 and IFNA16, which were elevated by chronic HIV infection. Elevated expression of HIV restriction factors was observed in chronically HIV-infected patients, which partly decreased during successful antiretroviral treatment. In vitro stimulation of peripheral blood mononuclear cells revealed that IFN-α6, -α14, and -α21 were most effective in inducing the expression of HIV restriction factors. These results indicate that HIV infection induces a specific expression pattern of IFN-α subtypes, which in turn induce the expression of various HIV restriction factors.

Characterization of the porcine alpha interferon multigene family.

The availability of data on the pig genome sequence prompted us to characterize the porcine IFN-alpha (PoIFN-alpha) multigene family. Fourteen functional PoIFN-alpha genes and two PoIFN-alpha pseudogenes were detected in the porcine genome. Multiple sequence alignment revealed a C-terminal deletion of eight residues in six subtypes. A phylogenetic tree of the porcine IFN-alpha gene family defined the evolutionary relationship of the various subtypes. In addition, analysis of the evolutionary rate and the effect of positive selection suggested that the C-terminal deletion is a strategy for preservation in the genome. Eight PoIFN-alpha subtypes were isolated from the porcine liver genome and expressed in BHK-21 cells line. We detected the level of transcription by real-time quantitative RT-PCR analysis. The antiviral activities of the products were determined by WISH cells/Vesicular Stomatitis Virus (VSV) and PK 15 cells/Pseudorabies Virus (PRV) respectively. We found the antiviral activities of intact PoIFN-alpha genes are approximately 2-50 times higher than those of the subtypes with C-terminal deletions in WISH cells and 15-55 times higher in PK 15 cells. There was no obvious difference between the subtypes with and without C-terminal deletion on acid susceptibility.

Role of p53 in the inhibitory effects of interferon-alpha subtypes on proliferation of hepatocellular carcinoma cells.

While interferon-alpha (IFN-alpha) subtypes share a common specific receptor composed of two subunits, interferon-alpha receptor (IFNAR)-1 and IFNAR-2, their subtype activities are exhibited via several intracellular signaling pathways and thus subsequently show different biological effects. Anti-proliferative effects of single treatment with IFN-alpha subtypes or 5-fluorouracil (FU), and of combined treatment with each IFN-alpha subtype and 5-FU were examined on three hepatocellular carcinoma cell lines, HepG2, HLE and PLC/PRF/5. HepG2 and PLC/PRF/5 cells were susceptible to the combination treatment, but HLE cells were not. Proliferation of PLC/PRF/5 cells was also inhibited by the IFN-alpha subtypes singly. In addition, apoptosis was observed in HepG2 cells upon treatment with 5-FU alone and with the combination treatment, and in PLC/PRF/5 cells after single treatment with the IFN-alpha subtypes and after the combination treatment. IFN-alpha subtypes induced cell cycle arrest in the G2/M phase in HepG2 and PLC/PRF/5. Analyses by Western blotting and immunoprecipitation revealed increased p53 phosphorylation in HepG2 and PLC/PRF/5 cells but not in HLE cells after combined treatment. Single treatment with IFN-alpha subtypes promoted p53 activation only in PLC/PRF/5 cells. These results propose that IFN-alpha subtypes induce cells to undergo apoptosis through p53 activation directly and indirectly, in collaboration with 5-FU, further suggesting the presence of distinct signal pathways for IFN-alpha-induced apoptosis.

Interferon-alpha hybrids.

The alpha-interferons (IFN-alpha) belong to a family of polypeptides comprising several subtypes. Using recombinant DNA technology, it has been possible to create IFN hybrids that provide novel combinations of the amino acid residues from the parental protein sequences. They have been used to study structure-activity relationships of IFN-alpha and interactions with the IFN-alpha receptor, and to create analogs of natural IFNs with novel properties for potential therapeutic application. The biological data obtained with these hybrids are now evaluated in terms of the published structural and homology models of IFN-beta and -alpha.

Variation in interferon sensitivity and induction between Usutu and West Nile (lineages 1 and 2) viruses.

Given the pivotal role of monocyte-derived dendritic cells (DCs) in determining the magnitude of the antiviral innate immune response, we sought to determine whether Usutu virus (USUV) and West Nile virus (WNV) lineages (L)1 and L2 can infect DCs and affect the rate of type I interferon (IFN) activation. The sensitivity of these viruses to types I and III IFNs was also compared. We found that USUV can infect DCs, induce higher antiviral activities, IFN alpha subtypes and the IFN stimulated gene (ISG)15 pathway, and is more sensitive to types I and III IFNs than WNVs. In contrast, we confirmed that IFN alpha/beta subtypes were more effective against WNV L2 than WNV L1. However, the replication kinetics, induction of IFN alpha subtypes and ISGs in DCs and the sensitivity to IFN lambda 1-3 did not differ between WNV L1 and L2.

In vivo expression of an interferon-alpha gene by intramuscular injection of naked DNA.

Acid-stable type I interferons belong to a multigene family. The biologic relevance of each subtype in vivo remains unknown. We have developed an experimental model in which muscles were transfected in situ with naked DNA plasmids encoding an IFN transgene to assess the roles of individual IFN subtypes in vivo. Murine IFN-alpha 9 gene was subcloned into several mammalian expression vectors. Adult C57BL/6 mice were injected bilaterally in regenerating tibialis anterior muscles with naked DNA 5 days after muscle injury to enhance DNA uptake and expression. In the muscles of mice given the IFN-alpha 9 plasmid constructs, acid-stable IFNs were detected by bioassay using reduction in cytopathic effect of encephalomyocarditis virus-infected L929 cells. In these same muscles, IFN-alpha 9 transcripts were identified by RT-PCR, indicating that transcription had occurred. Acid-stable IFNs were detected from days 7 to 28 post-DNA inoculation. Furthermore, these proteins were found in the sera of DNA-inoculated mice. Control groups of mice given the blank expression vectors did not produce detectable IFNs in muscle or sera as determined by bioassay, nor were transcripts detected by RT-PCR. This approach now allows investigation of the effector function of individual subtypes in various murine disease models.

Biologic and binding activities of IFN-alpha subtypes in ACHN human renal cell carcinoma cells and Daudi Burkitt's lymphoma cells.

Nine interferon-alpha subtypes, IFN-alpha1, IFN-alpha2, IFN-alpha5, IFN-alpha7, IFN-alpha8, IFN-alpha10, IFN-alpha14, IFN-alpha17, and IFN-alpha21, were separated from purified human lymphoblastoid IFN. We tested their inhibitory effects on cell growth and replication of Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV) and their induction of 2',5'-oligoadenylate synthetase (2', 5'-OAS) in ACHN renal cell carcinoma cells. In terms of all three activities, the nine subtypes had similar relative activities, with IFN-alpha10 the most active and IFN-alpha1 the least. Their relative effects on cell growth were similar in two other human cell lines, SK-LU-1 lung cancer cells and KU-2 renal cell carcinoma cells, whereas cells of the Daudi Burkitt lymphoma line behaved quite differently, being highly sensitive to all the nine subtypes. The relative effects with ACHN cells correlated well with their relative binding affinities. However, each of the subtypes bound to both ACHN and Daudi cells to almost the same extent. This suggests that their profound inhibitory effects on the growth of Daudi cells are amplified at some stage in the signal transduction pathway or in the expression of genes that results from binding to the IFN-alpha receptor.

Differential expression of IFN-alpha subtypes in human PBMC: evaluation of novel real-time PCR assays.

Studies of the human IFN-alpha subtype system have been hampered by the lack of efficient procedures to quantify and differentiate the expression of the highly homologous IFN-alpha subtypes. Here we evaluate four novel real-time PCR assays for the specific detection and quantification of IFN-alpha mRNA for the subtypes alpha(2), alpha(6), alpha(8) and alpha(1/13) in a combined assay in human peripheral blood mononuclear cells (PBMC). This included (a) the selection of beta-glucuronidase (GUS) as a suitable housekeeping gene for relative quantification; (b) verification of the specificity by using human DNA of different IFN-alpha subtypes; and (c) comparison of the amplification efficiencies among the different assays. This highly sensitive method allows the detection of low-level, constitutive IFN-alpha mRNA and shows differences in the composition of constitutive IFN-alpha subtypes compared to other cell types (HeLa and HEp-2). The in vitro stimulation of PBMC with Newcastle disease virus (NDV), Respiratory syncytial virus (RSV) or an inactivated Herpes simplex (HSV) preparation leads to the transcriptional induction of all IFN-alpha subtypes investigated but to different expression levels. Among the subtypes detected, IFN-alpha(13/1) and alpha(2) are the major transcripts followed by alpha(8), and finally alpha(6) as a minor transcribed subtype. Time-kinetics of IFN-alpha transcriptional activation also revealed variations in the course of IFN-alpha transcription between NDV, RSV or HSV. The data obtained from the real-time PCR assays correlated well with IFN-alpha(2) protein release. In conclusion, we have demonstrated the suitability and reliability of new real-time PCR assays for the rapid and efficient analysis of IFN-alpha subtype expression.