Poxviral B1 kinase overcomes barrier to autointegration factor, a host defense against virus replication

Barrier to autointegration factor (BAF) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. Herein, we demonstrate a cytoplasmic role for BAF in host defense during poxviral infections. Vaccinia is the prototypic poxvirus, a family of DNA viruses that replicate exclusively in the cytoplasm of infected cells. Mutations in the vaccinia B1 kinase (B1) compromise viral DNA replication, but the mechanism by which B1 achieves this has remained elusive. We now show that BAF acts as a potent inhibitor of poxvirus replication unless its DNA-binding activity is blocked by B1-mediated phosphorylation. These data position BAF as the effector of an innate immune response that prevents replication of exogenous viral DNA in the cytoplasm. To enable the virus to evade this defense, the poxviral B1 has evolved to usurp a signaling pathway employed by the host cell.     

IL-23 enhances host defense against vaccinia virus infection via a mechanism partly involving IL-17

To investigate roles of IL-23 in viral infection, we have engineered recombinant vaccinia virus (VV) expressing IL-12 (VV-IL-12) and expressing IL-23 (VV-IL-23). We found VV-IL-23 was less virulent in BALB/c mice than wild-type VV (VV-WT), indicating that IL-23 enhances resistance to VV. VV-specific CTL activity in VV-IL-23-infected mice was slightly higher than activity in VV-WT-inoculated mice, although antiviral Ab production and NK activity were not increased. IL-12/23p40-deficient mice survived the infection with VV-IL-23, indicating that IL-23 promotes VV resistance independently of IL-12. The mechanism of the IL-23-mediated resistance was distinct from that of the IL-12-regulated resistance because IFN-gamma-deficient mice did not eliminate VV-IL-12, but did eradicate VV-IL-23. These data indicate that IFN-gamma is essential for the IL-12-mediated resistance, but dispensable for the IL-23-regulated resistance. Because IL-17 is a key in the IL-23-regulated resistance to bacteria, we hypothesized an involvement of IL-17 in the resistance to VV. Treatment with an anti-IL-17 mAb resulted in a significant increase of viral titers in VV-IL-23-infected IFN-gamma-deficient mice. In addition, VV-IL-17 was less virulent than VV-WT in BALB/c mice, and IL-17-deficient mice were more sensitive to VV-WT than control mice. However, the effect of neutralization with an anti-IL-17 mAb was limited, and IL-17-deficient mice survived the infection with VV-IL-23. Taken together, these data suggest that the IL-23/IL-17 axis plays a certain but subdominant role in the IL-23-mediated resistance to VV. Unveiling of an alternative pathway in the IL-23-regulated resistance might provide a novel strategy against infectious pathogens without side effects of autoimmunity.     

Purification and characterization of vaccinia virus growth factor

A growth factor detected in the medium of vaccinia-virus-infected cells was purified to homogeneity. Sequence analysis shows it to be a processed form of a polypeptide encoded in the vaccinia virus genome which is related to epidermal growth factor (EGF) and alpha-transforming growth factor (alpha TGF). The amino terminus of the processed vaccinia virus growth factor (VVGF) begins at residue 20 of the primary translation product, indicating a signal sequence has been removed. The amino acid composition of purified VVGF predicts the carboxyl terminus is at, or near, residue 96, suggesting a transmembrane sequence has also been removed from secreted VVGF, which is, therefore, approximately 77 amino acids. VVGF, unlike EGF or alpha TGF, is glycosylated. VVGF binds to the EGF receptor and stimulates its autophosphorylation, suggesting that vaccinia virus has acquired sequences encoding a growth factor that may allow it to subvert EGF receptor-dependent functions.     

EB病毒免疫逃逸的分子机制

EB病毒(Epstein-Barr vius,EBV)是一种最广泛的对人类感染的γ疱疹病毒,与人类多种疾病尤其是恶性肿瘤有关。其致病的一个重要条件是能够在人体B细胞中长期潜伏,并且在人体免疫力低下时被激活并增殖,这表明EB病毒存在逃逸宿主细胞免疫的机制。从潜伏期EB病毒基因表达的下调、干扰抗原加工和提呈、调节细胞毒性T细胞(Cytotoxic lymphocyte,CTL)免疫应答、干扰细胞因子的作用、干扰CTL的活动及抑制宿主细胞凋亡、抑制辅助性T细胞1(Helper T cell 1,Th1)免疫应答等方面,对EB病毒免疫逃逸的分子机制作一简要综述。     

In vivo activity of lymphokine-activated macrophages in host defense against neoplasia

Purified splenic macrophage (M phi) from normal DBA/2J mice and mice bearing P815 tumors were examined for responsiveness to lymphokine (LK) preparations containing high concentrations of IFN-gamma. For both normal and tumor-bearing M phi, LK treatment induced morphologic changes and increased the percentage of Ia+ cells from 35 to 55%. Although neither population exhibited spontaneous cytotoxicity toward P815 targets, LK treatment induced considerable tumoricidal activity in tumor-bearing M phi (32 to 80% lysis) but only minimal activity in normal M phi (8 to 17% lysis). Subcutaneous injection of 1 X 10(6)P815 cells into DBA/2J led to progressive tumor growth and death of 100% of the recipients after 27 +/- 3 days. Injection of a 1:18 mixture of P815 with either LK-activated normal or tumor-bearing M phi caused tumor regression after 10 days, and prolonged life until 43 +/- 4 days with tumor-bearing M phi and 39 +/- 3 days with normal M phi. Untreated normal or tumor-bearing M phi were unable to cause the effect (30 +/- 2 days), and lymphocytes could not be substituted for M phi (25 +/- 3 days). In x-irradiated recipients, no effect of LK-activated M phi could be observed (control = 19 +/- 2 days; LK-activated tumor-bearing M phi = 21 +/- 3 days). In addition, administration of an admixture of LK-treated M phi and x-rayed tumor before challenge with viable P815 enabled the recipient to inhibit tumor growth and caused tumor necrosis with inflammatory cell infiltration of the tumor. These observations suggest that, in part, LK-activated M phi may interact in vivo with host-derived cellular components and enhance the immune reactivity of the host against the tumor.     

How vaccinia virus has evolved to subvert the host immune response

Viruses are obligate intracellular parasites and are some of the most rapidly evolving and diverse pathogens encountered by the host immune system. Large complicated viruses, such as poxviruses, have evolved a plethora of proteins to disrupt host immune signalling in their battle against immune surveillance. Recent X-ray crystallographic analysis of these viral immunomodulators has helped form an emerging picture of the molecular details of virus-host interactions. In this review we consider some of these immune evasion strategies as they apply to poxviruses, from a structural perspective, with specific examples from the European SPINE2-Complexes initiative. Structures of poxvirus immunomodulators reveal the capacity of viruses to mimic and compete against the host immune system, using a diverse range of structural folds that are unique or acquired from their hosts with both enhanced and unexpectedly divergent functions.     

Protective role of beta interferon in host defense against influenza A virus

Type I interferon (IFN), which includes the IFN-alpha and -beta subtypes, plays an essential role in host defense against influenza A virus. However, the relative contribution of IFN-beta remains unresolved. In mice, type I IFN is effective against influenza viruses only if the IFN-induced resistance factor Mx1 is present, though most inbred mouse strains, including the recently developed IFN-beta-deficient mice, bear only defective Mx1 alleles. We therefore generated IFN-beta-deficient mice carrying functional Mx1 alleles (designated Mx-BKO) and compared them to either wild-type mice bearing functional copies of both IFN-beta and Mx1 (designated Mx-wt) or mice carrying functional Mx1 alleles but lacking functional type I IFN receptors (designated Mx-IFNAR). Influenza A virus strain SC35M (H7N7) grew to high titers and readily formed plaques in monolayers of Mx-BKO and Mx-IFNAR embryo fibroblasts which showed no spontaneous expression of Mx1. In contrast, Mx-wt embryo fibroblasts were found to constitutively express Mx1, most likely explaining why SC35M did not grow to high titers and formed no visible plaques in such cells. In vivo challenge experiments in which SC35M was applied via the intranasal route showed that the 50% lethal dose was about 20-fold lower in Mx-BKO mice than in Mx-wt mice and that virus titers in the lungs were increased in Mx-BKO mice. The resistance of Mx-BKO mice to influenza A virus strain PR/8/34 (H1N1) was also substantially reduced, demonstrating that IFN-beta plays an important role in the defense against influenza A virus that cannot be compensated for by IFN-alpha.     

Molecular attenuation of vaccinia virus: mutant generation and animal characterization.

These studies demonstrated that the inbred BALB/c mouse strain can be optimized for the assessment of vaccinia virus virulence, growth, and spread from the site of inoculation and immune protection from a lethal vaccinia virus challenge. The studies established that manipulation of the vaccinia virus genome generated mutants exhibiting a wide range of attenuated phenotypes. The nine NYCBH vaccinia virus mutants had intracranial 50% lethal doses that ranged from 2 to greater than 7 log10 units. The decreased neurovirulence was due to decreased replication in brain tissue. Three mutants had a decreased ability to disseminate to the lungs, brains, livers, and spleens of mice after intranasal infection. One mutant had a decreased transmission from mice infected by tail scarification to naive cage mates. Although the mutants, with one exception, grew to wild-type titers in cell culture, they showed a growth potential on the scarified skin of mice that was dramatically different from that of the wild-type virus. Consequently, all of the mutants had significantly compromised immunogenicities at low virus immunization doses compared with that of the wild-type virus. Conversely, at high immunization doses most mutants could induce an immune response similar to that of the wild-type virus. Three Wyeth vaccine strain mutants were also studied. Whereas the thymidine kinase, ribonucleotide reductase, and hemagglutinin mutants had a reduced virulence (50% lethal dose), only the thymidine kinase mutant retained its immunogenicity.     

Involvement of the platelet-activating factor receptor in host defense against Streptococcus pneumoniae during postinfluenza pneumonia

Although influenza infection alone may lead to pneumonia, secondary bacterial infections are a much more common cause of pneumonia. Streptococcus pneumoniae is the most frequently isolated causative pathogen during postinfluenza pneumonia. Considering that S. pneumoniae utilizes the platelet-activating factor receptor (PAFR) to invade the respiratory epithelium and that the PAFR is upregulated during viral infection, we here used PAFR gene-deficient (PAFR-/-) mice to determine the role of this receptor during postinfluenza pneumococcal pneumonia. Viral clearance was similar in wild-type and PAFR-/- mice, and influenza virus was completely removed from the lungs at the time mice were inoculated with S. pneumoniae (day 14 after influenza infection). PAFR-/- mice displayed a significantly reduced bacterial outgrowth in their lungs, a diminished dissemination of the infection, and a prolonged survival. Pulmonary levels of IL-10 and KC were significantly lower in PAFR-/- mice, whereas IL-6 and TNF-alpha were only trendwise lower. These data indicate that the pneumococcus uses the PAFR leading to severe pneumonia in a host previously exposed to influenza A.     

Role of Nrf2 in host defense against influenza virus in cigarette smoke-exposed mice

Influenza virus is a common respiratory tract viral infection. Although influenza can be fatal in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, its pathogenesis is not fully understood. The Nrf2-mediated antioxidant system is essential to protect the lungs from oxidative injury and inflammation. In the present study, we investigated the role of Nrf2 in protection against influenza virus-induced pulmonary inflammation after cigarette smoke exposure with both in vitro and in vivo approaches. For in vitro analyses, peritoneal macrophages isolated from wild-type and Nrf2-deficient mice were treated with poly(I:C) and/or cigarette smoke extract. For in vivo analysis, these mice were infected with influenza A virus with or without exposure to cigarette smoke. In Nrf2-deficient macrophages, NF-κB activation and the induction of its target inflammatory genes were enhanced after costimulation with cigarette smoke extract and poly(I:C) compared with wild-type macrophages. The induction of antioxidant genes was observed for the lungs of wild-type mice but not those of Nrf2-deficient mice after cigarette smoke exposure. Cigarette smoke-exposed Nrf2-deficient mice showed higher rates of mortality than did wild-type mice after influenza virus infection, with enhanced peribronchial inflammation, lung permeability damage, and mucus hypersecretion. Lung oxidant levels and NF-κB-mediated inflammatory gene expression in the lungs were also enhanced in Nrf2-deficient mice. Our data indicate that the antioxidant pathway controlled by Nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and injury under oxidative conditions.     

Expression of the E3L gene of vaccinia virus in transgenic mice decreases host resistance to vaccinia virus and Leishmania major infections

The E3L gene of vaccinia virus (VACV) encodes the E3 protein that in cultured cells inhibits the activation of interferon (IFN)-induced proteins, double-stranded RNA-dependent protein kinase (PKR), 2′-5′-oligoadenylate synthetase/RNase L (2-5A system) and adenosine deaminase (ADAR-1), thus helping the virus to evade host responses. Here, we have characterized the in vivo E3 functions in a murine inducible cell culture system (E3L-TetOFF) and in transgenic mice (TgE3L). Inducible E3 expression in cultured cells conferred on cells resistance to the antiviral action of IFN against different viruses, while expression of the E3L gene in TgE3L mice triggered enhanced sensitivity of the animals to pathogens. Virus infection monitored in TgE3L mice by different inoculation routes (intraperitoneal and tail scarification) showed that transgenic mice became more susceptible to VACV infection than control mice. TgE3L mice were also more susceptible to Leishmania major infection, leading to an increase in parasitemia compared to control mice. The enhanced sensitivity of TgE3L mice to VACV and L. major infections occurred together with alterations in the host immune system, as revealed by decreased T-cell responses to viral antigens in the spleen and lymph nodes and by differences in the levels of specific innate cell populations. These results demonstrate that expression of the E3L gene in transgenic mice partly reverses the resistance of the host to viral and parasitic infections and that these effects are associated with immune alterations.     

Moxibustion activates host defense against herpes simplex virus type I through augmentation of cytokine production

Moxibustion is a technique used in traditional oriental medicine, the aim of which is to cure and/or prevent illness by activating a person's ability for self‐healing. In this study, we assessed how moxibustion would affect the immune system and whether it would augment protective immunity. Mice were treated with moxibustion at Zusanli (ST36) acupoints; we analyzed mortality and cytokine activity in sera after infection with herpes simplex virus type 1 (HSV‐1), and cytokine gene expression in the skin and the spleen without a virus challenge. Our study demonstrates that pretreatment of BALB/c mice with moxibustion resulted in a marked increase in the survival rate after infection with lethal doses of HSV‐1, and elevated serum levels of IL‐1β and IFN‐γ on days 1 and 6 post‐infection with HSV‐1. Semi‐quantitative RT‐PCR assay showed that moxibustion treatment augmented the expression of IL‐1α, IL‐1β, IL‐6, universal‐IFN‐α, MIP‐1α, and TNF‐α mRNA in the skin, and IL‐1α, IL‐1β, IL‐12p40, IL‐15, u‐IFN‐α, MIP‐1α, and TNF‐α mRNA in the spleen. Moreover, moxibustion induces augmentation of natural killer cell activity. Collectively, our study demonstrates that moxibustion activates protective responses against HSV‐1 infection through the activation of cytokine production including IFN, and of NK cells.     

Isolation and characterization of neutralizing monoclonal antibodies to vaccinia virus.

Cells producing neutralizing monoclonal antibodies (mAbs) to UV-inactivated vaccinia virus strain WR were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. Three mAbs that reacted strongly with purified virus envelopes as determined by enzyme-linked immunosorbent assay were studied. The three mAbs recognized a 14,000-molecular-weight (14K) envelope protein of vaccinia virus and were shown to be immunoglobulin G2b (mAbC3 and mAbB11) and immunoglobulin M (mAbF11). By using ascites, one of the antibodies, mAbC3, neutralized (50%) virus infectivity with a titer of about 10(-4), whereas the others exhibited lower neutralization titers of 10(-2) to 10(-3). The binding of the mAbs to vaccinia virus did not alter virus attachment to cells. However, virus uncoating was extensively blocked by mAbC3, whereas mAbB11 and mAbF11 had little or no effect. The three mAbs recognized a similar 14K protein in cowpox, rabbitpox, and vaccinia Elstree strains, indicating a high degree of protein conservation among orthopoxviruses. Based on the binding of mAbs to V-8 protease cleavage products of the 14K protein, the extent of protein recognition for other poxviruses, and differences in the degree of virus neutralization and of virus uncoating into cells, we suggest that the three mAbs recognize different domains of vaccinia 14K viral envelope protein. Furthermore, our findings indicate that the 14K protein may play a role in virus penetration.     

Age-dependent role for CCR5 in antiviral host defense against herpes simplex virus type 2

Elimination of viral infections is dependent on rapid recruitment and activation of leukocytes with antiviral activities to infected areas. Chemokines constitute a class of cytokines that have regulatory effects on leukocyte migration and activity. In this study we have studied the role of CC chemokine receptor 1 (CCR1) and CCR5 in host defense during a generalized herpes simplex virus type 2 (HSV-2) infection. Whereas both 4- and 8-week-old CCR1(-/-) mice resembled wild-type mice (C57BL/6) with respect to defense against the infection, significantly higher virus titers were seen in the livers and brains of 4-week-old CCR5(-/-) mice. At the age of 8 weeks, CCR5(-/-) were indistinguishable from wild-type mice and cleared the infection from liver and spleen. Although 4-week-old CCR5(-/-) mice were able to recruit natural killer (NK) cells to the site of infection, these cells had reduced cytotoxic activity compared to NK cells from wild-type mice. This was not due to lower production of alpha/beta interferon or interleukin-12, two well-described activators of cytotoxic activity in NK cells. We also noted that the spleens of young CCR5(-/-) mice did not increase in size during infection as did the spleens of wild-type and CCR1(-/-) mice. This observation was accompanied by impaired proliferation of CCR5(-/-) splenocytes (SCs) ex vivo. Moreover, migration of CD8(+) T cells to the liver in response to infection was impaired in CCR5(-/-) mice, and adoptive transfer of SCs from CCR5(-/-) mice infected for 6 days into newly infected wild-type mice did not improve antiviral activity in the liver, in contrast to what was seen in mice receiving immune SCs from wild-type mice. Altogether, this study shows that CCR5 plays an age-dependent role in host defense against HSV-2 by supporting both the innate and adaptive immune response.     

Deletion of the vaccinia virus growth factor gene reduces virus virulence.

The vaccinia virus growth factor (VGF) gene encodes a polypeptide with amino acid sequence homology to epidermal growth factor (EGF) and transforming growth factor alpha and is present twice, once at each end of the virus genome within the inverted terminal repetition. Recombination procedures were used to replace more than half of both VGF genes with a beta-galactosidase cassette which served as a color indicator for isolating an unconditionally viable VGF- mutant. The VGF- mutant genotype and phenotype were confirmed by Southern blot analysis and assays for functional growth factor. The plaque-forming efficiencies of VGF- and wild-type (WT) viruses were similar in a variety of cell types containing low or high densities of EGF receptors, suggesting a lack of a specific requirement for either VGF or the EGF receptor in the initiation of virus infection. The yield of VGF- virus was similar to that of WT virus in growing BS-C-1 and Swiss 3T3 cells, but lower in resting Swiss 3T3 cells. The greatest differences between VGF- and WT virus occurred in vivo: higher doses of VGF- virus than WT virus were required for intracranial lethality in mice and for production of skin lesions in rabbits. Thus, expression of the VGF gene is important to the virulence of vaccinia virus.     

Insights from human studies into the host defense against candidiasis

Candida spp. are the most common cause of mucosal and disseminated fungal infections in humans. Studies using mutant strains of mice have provided initial information about the roles of dectin-1, CARD9, and Th17 cytokines in the host defense against candidiasis. Recent technological advances have resulted in the identification of mutations in specific genes that predispose humans to develop candidal infection. The analysis of individuals with these mutations demonstrates that dectin-1 is critical for the host defense against vulvovaginal candidiasis and candidal colonization of the gastrointestinal tract. They also indicate that CARD9 is important for preventing both mucosal and disseminated candidiasis, whereas the Th17 response is necessary for the defense against mucocutaneous candidiasis. This article reviews the recent studies of genetic defects in humans that result in an increased susceptibility to candidiasis and discusses how these studies provide new insight into the host defense against different types of candidal infections.     

Antiviral activity and increased host defense against influenza infection elicited by the human cathelicidin LL-37

The extensive world-wide morbidity and mortality caused by influenza A viruses highlights the need for new insights into the host immune response and novel treatment approaches. Cationic Host Defense Peptides (CHDP, also known as antimicrobial peptides), which include cathelicidins and defensins, are key components of the innate immune system that are upregulated during infection and inflammation. Cathelicidins have immunomodulatory and anti-viral effects, but their impact on influenza virus infection has not been previously assessed. We therefore evaluated the effect of cathelicidin peptides on disease caused by influenza A virus in mice. The human cathelicidin, LL-37, and the murine cathelicidin, mCRAMP, demonstrated significant anti-viral activity in vivo, reducing disease severity and viral replication in infected mice to a similar extent as the well-characterized influenza virus-specific antiviral drug zanamivir. In vitro and in vivo experiments suggested that the peptides may act directly on the influenza virion rather than via receptor-based mechanisms. Influenza virus-infected mice treated with LL-37 had lower concentrations of pro-inflammatory cytokines in the lung than did infected animals that had not been treated with cathelicidin peptides. These data suggest that treatment of influenza-infected individuals with cathelicidin-derived therapeutics, or modulation of endogenous cathelicidin production may provide significant protection against disease.