Mouse hepatitis coronavirus A59 nucleocapsid protein is a type I interferon antagonist

The recent emergence of several new coronaviruses, including the etiological cause of severe acute respiratory syndrome, has significantly increased the importance of understanding virus-host cell interactions of this virus family. We used mouse hepatitis virus (MHV) A59 as a model to gain insight into how coronaviruses affect the type I alpha/beta interferon (IFN) system. We demonstrate that MHV is resistant to type I IFN. Protein kinase R (PKR) and the alpha subunit of eukaryotic translation initiation factor are not phosphorylated in infected cells. The RNase L activity associated with 2',5'-oligoadenylate synthetase is not activated or is blocked, since cellular RNA is not degraded. These results are consistent with lack of protein translation shutoff early following infection. We used a well-established recombinant vaccinia virus (VV)-based expression system that lacks the viral IFN antagonist E3L to screen viral genes for their ability to rescue the IFN sensitivity of the mutant. The nucleocapsid (N) gene rescued VVDeltaE3L from IFN sensitivity. N gene expression prevents cellular RNA degradation and partially rescues the dramatic translation shutoff characteristic of the VVDeltaE3L virus. However, it does not prevent PKR phosphorylation. The results indicate that the MHV N protein is a type I IFN antagonist that likely plays a role in circumventing the innate immune response.     

Endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry

Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.     

Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.     

Modulation of the type I interferon pathways by culture-adaptive hepatitis C virus core mutants

Hepatitis C virus (HCV) often establishes a persistent infection that leads to chronic liver diseases. The viral core protein modulates various cellular activities involved in this process. We found two mutations, K23E and V31A, in the core gene of the transfected HCV JFH-1 genome, which had been replicated for a prolonged period. The mutant viruses escaped immunochemical detection by a core-specific antibody and demonstrated enhanced RNA replication and protein expression, compared to the parental virus. The mutant core proteins bound less tightly than the parental type core to the DEAD-box RNA helicase DDX3 and attenuated the TBK1-mediated activation of interferon-related promoters. These results suggest a mechanism by which the viruses adapt to attenuate cellular antiviral activity and to establish persistent infection.     

CpG-ODNs induces up-regulated expression of chemokine CCL9 in mouse macrophages and microglia

Unmethylated CpG oligodeoxynucleotides (CpG-ODNs) interact with Toll-like receptor (TLR) 9 to activate macrophage/microglia in central nervous system (CNS). Here, we investigated the potential involvement of the chemokine CCL9 and its receptor CCR1 in the effects of CpG-ODNs on macrophage/microglial cells. CpG-ODNs enhanced the expression of TLR9 mRNA of RAW264.7 macrophage and BV2 microglia cells time dependently. The expression of CCL9 of macrophages/microglia showed different responsiveness upon stimulation with a variety of CpG-ODN sequences. The CpG-ODNs-mediated induction of CCL9 was TLR9/MyD88 dependent and associated with activation of stress kinases, particularly ERK, p38 MAPK and PI3K. The expression of CCR1 was also significantly increased by CpG-ODNs that increased CCL9 expression. These results reveal the potential involvement of CCL9 and CCR1 in regulation of macrophage and microglial cells by CpG-ODNs and may help improving our understanding about the role of the chemokine/chemokine receptor pairs in macrophage/microglia under physiologic and pathologic conditions.     

Sensitivity of rabies virus to type I interferon is determined by the phosphoprotein gene

The growth of a virulent strain of fixed rabies virus, Nishigahara, in mouse neuroblastoma NA cells treated with type I interferon (IFN) was compared with that of a derivative avirulent strain, Ni-CE. Nishigahara strain was slightly sensitive to IFN treatment but still grew more efficiently than did Ni-CE strain in IFN-treated NA cells. Furthermore, a virulent chimeric virus with the phosphoprotein gene from Nishigahara strain in the Ni-CE genome was less sensitive to IFN treatment than was Ni-CE strain, indicating that the IFN sensitivity is determined by the phosphoprotein gene of the virus.     

RIG-I mediates the co-induction of tumor necrosis factor and type I interferon elicited by myxoma virus in primary human macrophages

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection.     

Cytomegalovirus induces interferon-stimulated gene expression and is attenuated by interferon in the developing brain

Cytomegalovirus (CMV) is considered the most common infectious agent causing permanent neurological dysfunction in the developing brain. We have previously shown that CMV infects developing brain cells more easily than it infects mature brain cells and that this preference is independent of the host B- and T-cell responses. In the present study, we examined the innate antiviral defenses against mouse (m) and human (h) CMVs in developing and mature brain and brain cells. mCMV infection induced interferon (IFN)-stimulated gene expression by 10- to 100-fold in both glia- and neuron-enriched cultures. Treatment of primary brain cultures with IFN-alpha, -beta, and -gamma or a synthetic RNA, poly(I:C), reduced the number of mCMV-infected cells, both in older cells and in fresh cultures from embryonic mouse brains. When a viral dose that killed almost all unprotected cells was used, IFN-protected cells had a natural appearance, and when they were tested with whole-cell patch clamp recording, they appeared physiologically normal with typical resting membrane potentials and action potentials. mCMV infection increased expression of representative IFN-stimulated genes (IFIT3, OAS, LMP2, TGTP, and USP18) in both neonatal and adult brains to similarly large degrees. The robust upregulation of gene expression in the neonatal brain was associated with a much higher degree of viral replication at this stage of development. In contrast to the case for downstream gene induction, CMV upregulated IFN-alpha/beta expression to a greater degree in the adult brain than in the neonatal brain. Similar to the case with cultured brain cells, IFN treatment of the developing brain in vivo depressed mCMV replication. In parallel work with cultured primary human brain cells, IFN and poly(I:C) treatment reduced hCMV infection and prevented virus-mediated cell death. These results suggest that coupling IFN administration with current treatments may reduce CMV infections in the developing brain.     

Differential type I interferon induction by respiratory syncytial virus and influenza a virus in vivo

Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-alpha/beta induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-alpha/beta production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-alpha/beta in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.     

Characterization of a variant virus selected in rat brains after infection by coronavirus mouse hepatitis virus JHM.

The intracerebral inoculation of Lewis rats with the murine coronavirus MHV-JHM leads in the majority of animals to acute encephalitis and death within 14 days. Viral RNAs isolated from the brains of animals 5 to 7 days after infection were compared by Northern blot analysis with the RNAs produced during the lytic infection of Sac(-) or DBT cells with wild-type MHV-JHM (wt virus). Reproducibly, the subgenomic mRNAs 2 and 3 but no other viral RNAs were significantly larger in the brain-derived material. All viruses isolated from infected brain material displayed and maintained this altered mRNA profile when cultivated in Sac(-) or DBT cells. A virus isolated from the infected brain material, MHV-JHM clone 2 (cl-2 virus), has been further characterized. This isolate grew in tissue culture and induced cytopathic effects comparable to those induced by wt virus. However, the mRNAs 2 and 3 produced in cl-2 virus-infected cells had molecular weights ca. 150,000 larger than those produced in cells infected with wt virus. There was no detectable difference in genome-sized RNA (mRNA 1) or subgenomic mRNAs 4, 5, 6, and 7 as determined by electrophoresis in agarose gels. T1-resistant oligonucleotide analysis of genomic RNA revealed one additional and one missing oligonucleotide in the fingerprint of cl-2 virus compared with wt virus. The oligonucleotide fingerprints of intracellular mRNA 3 were identical for both viruses. Pulse-labeling with [35S]methionine in the presence of tunicamycin showed that the primary translation product of mRNA 3, the E2 apoprotein, was ca. 15,000 larger in molecular weight in cl-2 virus-infected cells. These data show that viruses with larger mRNAs 2 and 3 (the latter encoding an altered E2 glycoprotein) are selected for multiplication in rat brains. Mechanisms for the generation of such variants and the possible nature of their selective advantage are considered.     

Ceacam1a-/- mice are completely resistant to infection by murine coronavirus mouse hepatitis virus A59

CEACAM1a glycoproteins are members of the immunoglobulin (Ig) superfamily and the carcinoembryonic antigen family. Isoforms expressing either two or four alternatively spliced Ig-like domains in mice have been found in a number of epithelial, endothelial, or hematopoietic tissues. CEACAM1a functions as an intercellular adhesion molecule, an angiogenic factor, and a tumor cell growth inhibitor. Moreover, the mouse and human CEACAM1a proteins are targets of viral or bacterial pathogens, respectively, including the murine coronavirus mouse hepatitis virus (MHV), Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, as well as Moraxella catarrhalis in humans. We have shown that targeted disruption of the Ceacam1a (MHVR) gene resulting in a partial ablation of the protein in mice (p/p mice) led to reduced susceptibility to MHV-A59 infection of the modified mice in the BALB/c background. We have now engineered and produced a Ceacam1a-/- mouse that exhibits complete ablation of the CEACAM1a protein in every tissue where it is normally expressed. We report that 3-week-old Ceacam1a-/- mice in the C57BL/6 genetic background are fully resistant to MHV-A59 infection by both intranasal and intracerebral routes. Whereas virus-inoculated wild-type +/+ C57BL/6 mice showed profound liver damage and spinal cord demyelination under these conditions, Ceacam1a-/- mice displayed normal livers and spinal cords. Virus was recovered from liver and spinal cord tissues of +/+ mice but not of -/- mice. These results indicate that CEACAM1a is the sole receptor for MHV-A59 in both liver and brain and that its deletion from the mouse renders the mouse completely resistant to infection by this virus.     

Lack of repeat transduction by recombinant adeno-associated virus type 5/5 vectors in the mouse airway

While recombinant adeno-associated virus (rAAV) vectors promote long-term transgene expression in the lungs and other organs, the goal of correcting chronic inherited lung diseases such as cystic fibrosis with this type of viral gene transfer vector is limited by the requirement of achieving stable potent transgene expression, potentially requiring vector readministration. Here we evaluated the abilities of rAAV type 5/5 (rAAV5/5) vectors based on the genome and capsid of AAV5 to efficiently transduce the lungs and nasal epithelium of mice after repeated administration. Transduction efficiency as judged by reporter gene expression was markedly reduced on a second rAAV5/5 administration and effectively abolished on a third. Varying the period between administrations from 8 to 36 weeks did not allow efficient repeated administration. A rapid rise in anti-AAV5 antibodies was noted after rAAV5/5 vector administration that was sustained for the entire period of investigation (in some cases exceeding 9 months). Furthermore, this antibody response and subsequent failure to repeatedly administer the vector were not rescued by the in vivo expression of CTLA4Ig from an rAAV5/5 vector. These results suggest that without the development of an effective and clinically acceptable immunosuppression strategy, treatments for chronic diseases that require repeated administration of rAAV5/5 vectors will be unsuccessful.     

Control of mouse hepatitis virus replication in macrophages by a recessive gene on chromosome 7

We have identified and mapped a new locus that imparts resistance to productive infection of mouse hepatitis virus MHV(A59) in cultured macrophages. This locus maps 41.5 centimorgans from the albino locus on chromosome 7.