IRF-3 activation by Sendai virus infection is required for cellular apoptosis and avoidance of persistence

Here, we report that specific manipulations of the cellular response to virus infection can cause prevention of apoptosis and consequent establishment of persistent infection. Infection of several human cell lines with Sendai virus (SeV) or human parainfluenza virus 3, two prototypic paramyxoviruses, caused slow apoptosis, which was markedly accelerated upon blocking the action of phosphatidylinositol 3-kinases (PI3 kinases) in the infected cells. The observed apoptosis required viral gene expression and the action of the caspase 8 pathway. Although virus infection activated PI3 kinase, as indicated by AKT activation, its blockage did not inhibit JNK activation or IRF-3 activation. The action of neither the Jak-STAT pathway nor the NF-kappaB pathway was required for apoptosis. In contrast, IRF-3 activation was essential, although induction of the proapototic protein TRAIL by IRF-3 was not required. When IRF-3 was absent or its activation by the RIG-I pathway was blocked, SeV established persistent infection, as documented by viral protein production and infectious virus production. Introduction of IRF-3 in the persistently infected cells restored the cells' ability to undergo apoptosis. These results demonstrated that in our model system, IRF-3 controlled the fate of the SeV-infected cells by promoting apoptosis and preventing persistence.     

The N- and C-terminal domains of the NS1 protein of influenza B virus can independently inhibit IRF-3 and beta interferon promoter activation

The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only approximately 20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of alpha/beta interferon (IFN-alpha/beta) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-alpha/beta synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-3 nuclear translocation and IFN-beta promoter activation. A mutational analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity correlated with IFN-beta promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation, as determined by its nuclear translocation, and of IFN-alpha/beta synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-3- and IFN-induced antiviral host responses to virus infection.     

Influence of H-2 complex on susceptibility to infection by murine leukemia virus.

Titers of infectious ecotropic MuLV in mouse spleen were examined after deliberate infection. In congenic mice differing only in H-2 haplotype, a gene (or genes) within the H-2 complex determined either a high virus titer (H-2k, H-2d, H-2a) or a low titer (H-2b, H-2q). Susceptibility to high virus titers was inherited as a dominant trait. Kinetic studies revealed similar initial patterns of infection in both groups, with a fall in titer in the "resistant" strain occurring from week 6 through 10 after infection. Anti-VEA antibody titers differed significantly between the groups, but no mechanistic role for antibody in eliminating virus was apparent. Genes outside the H-2 complex were shown to influence MuLV titers after infection as well.     

Distinct organ-dependent mechanisms for the control of murine cytomegalovirus infection by natural killer cells.

Antiviral mechanisms by which natural killer (NK) cells control murine cytomegalovirus (MCMV) infection in the spleens and livers of C57BL/6 mice were measured, revealing different mechanisms of control in different organs. Three days postinfection, MCMV titers in the spleens of perforin 0/0 mice were higher than in those of perforin +/+ mice, but no elevation of liver titers was found in perforin 0/0 mice. NK cell depletion in MCMV-infected perforin 0/0 mice resulted only in an increase in liver viral titers and not in spleen titers. Depletion of gamma interferon (IFN-gamma) in C57BL/6 mice by injections with monoclonal antibodies to IFN-gamma resulted in an increase of viral titers in the liver but not in the spleen. Analyses using IFN-gamma-receptor-deficient mice, rendered chimeric with C57BL/6 bone marrow cells, indicated that in a recipient environment where IFN-gamma cannot exert its effects, the depletion of NK cells caused an increase in MCMV titers in the spleens but had little effect in the liver. IFN-gamma has the ability to induce a variety of cells to produce nitric oxide, and administrating the nitric oxide synthase inhibitor N(omega)-monomethyl-L-arginine into MCMV-infected C57BL/6 mice resulted in MCMV titer increases in the liver but not in the spleen. Taken together, these data suggest that in C57BL/6 mice, there is a dichotomy in the mechanisms utilized by NK cells in the regulation of MCMV in different organs. In the spleen NK cells exert their effects in a perforin-dependent manner, suggesting a cytotoxic mechanism, while in the liver the production of IFN-gamma by NK cells may be a predominant mechanism in the regulation of MCMV synthesis. These results may explain why the Cmv-lr locus, which maps closely to genes regulating NK cell cytotoxic function, confers an NK cell-dependent resistance to MCMV infection in the spleen but not in the liver.     

Interferon-alpha suppresses leptin levels: studies in interferon-alpha treated patients with hepatitis C virus infection and murine adipocytes

Leptin is an adipocyte-derived protein, which signals the status of current energy stores and energy intake to the central nervous system. Interferon-alpha therapy is frequently associated with loss of appetite and weight reduction. In this study, we tested whether interferon-alpha is able to regulate leptin synthesis. We therefore determined leptin plasma concentrations in thirteen patients with chronic hepatitis C who were treated with 1 x 10(7) IU interferon-alpha daily, up to 21 days after initial treatment. Furthermore, leptin concentrations and messenger RNA levels were quantified in interferon-alpha-treated and untreated murine adipocytes. After reaching peak plasma levels at 12 hours, probably reflecting the circadian rhythm, leptin concentrations fell and were significantly lower after 14 days. They remained significantly decreased 17 and 21 days after the start of the interferon-alpha treatment. In murine adipocytes exposed to interferon-alpha, leptin secretion was significantly decreased while messenger RNA levels remained unchanged. Our data suggest that, in contrast to proinflammatory cytokines such as interleukin-1, interferon-alpha suppresses leptin secretion in adipose tissue. We therefore hypothesize that loss of weight and appetite in interferon-alpha-treated patients with chronic hepatitis C might not be due to an elevation of leptin resulting in signals of increased energy stores in the brain.     

Up-Regulation of Intestinal Epithelial Cell Derived IL-7 Expression by Keratinocyte Growth Factor through STAT1/IRF-1, IRF-2 Pathway

Background

Epithelial cells(EC)-derived interleukin-7 (IL-7) plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL), and keratinocyte growth factor (KGF) exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine.

Methods

Intestinal epithelial cells (LoVo cells) and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA).

Results

KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively.

Conclusion

KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system.     

Target cell heterogeneity in murine leukemia virus infection. III. Identification of susceptible Lyt 1+ and resistant Lyt 2+ T-cell subsets following in vitro infection with Friend murine leukemia virus

The susceptibility of splenic T-cell subpopulations to productive infection with Friend murine leukemia virus was determined after in vitro infection and stimulation with Con A. Con A enhanced the number of productively infected cells in unseparated spleen cells as well as in T-cell-enriched spleen cell fractions. Splenic T cells were fractionated into Lyt 1+ and Lyt 2+ subpopulations using both positive and negative selection techniques; susceptible splenic T cells were recovered in the Lyt 1+ fraction and specific cytotoxic treatment with anti-Lyt 1 antibody and complement reduced the number of infectious center-producing cells by greater than 87%. In marked contrast, Lyt 2+ splenic T cells were resistant to productive infection by Friend murine leukemia virus in vitro.