Dengue virus (DV) infection is one of the most common mosquito-borne viral diseases in the world. The innate immune system is important for the early detection of virus and for mounting a cascade of defense measures which include the production of type 1 interferon (IFN). Hence, a thorough understanding of the innate immune response during DV infection would be essential for our understanding of the DV pathogenesis. A recent application of the microarray to dengue virus type 1 (DV1) infected lung carcinoma cells revealed the increased expression of both extracellular and cytoplasmic pattern recognition receptors; retinoic acid inducible gene-I (RIG-I), melanoma differentiation associated gene-5 (MDA-5) and Toll-like receptor-3 (TLR3). These intracellular RNA sensors were previously reported to sense DV infection in different cells. In this study, we show that they are collectively involved in initiating an effective IFN production against DV. Cells silenced for these genes were highly susceptible to DV infection. RIG-I and MDA5 knockdown HUH-7 cells and TLR3 knockout macrophages were highly susceptible to DV infection. When cells were silenced for only RIG-I and MDA5 (but not TLR3), substantial production of IFN-β was observed upon virus infection and vice versa. High susceptibility to virus infection led to ER-stress induced apoptosis in HUH-7 cells. Collectively, our studies demonstrate that the intracellular RNA virus sensors (RIG-I, MDA5 and TLR3) are activated upon DV infection and are essential for host defense against the virus.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is an inefficient inducer of interferon (IFN) response. It expresses various proteins that effectively circumvent IFN production at different levels via distinct mechanisms. Through the construction of recombinant IBV expressing proteins 8a, 8b and 8ab encoded by SARS-CoV ORF8, we demonstrate that expression of 8b and 8ab enables the corresponding recombinant viruses to partially overcome the inhibitory actions of IFN activation to achieve higher replication efficiencies in cells. We also found that proteins 8b and 8ab could physically interact with IRF3. Overexpression of 8b and 8ab resulted in the reduction of poly (I:C)-induced IRF3 dimerization and inhibition of the IFN-β signaling pathway. This counteracting effect was partially mediated by protein 8b/8ab-induced degradation of IRF3 in a ubiquitin-proteasome-dependent manner. Taken together, we propose that SARS-CoV may exploit the unique functions of proteins 8b and 8ab as novel mechanisms to overcome the effect of IFN response during virus infection.

The most striking difference between the subgenomic mRNA8 of severe acute respiratory syndrome coronavirus isolated from human and some animal species is the deletion of 29 nucleotides, resulting in splitting of a single ORF (ORF8) into two ORFs (ORF8a and ORF8b). ORF8a and ORF8b are predicted to encode two small proteins, 8a and 8b, and ORF8 a single protein, 8ab (a fusion form of 8a and 8b). To understand the functions of these proteins, we cloned cDNA fragments covering these ORFs into expression plasmids, and expressed the constructs in both in vitro and in vivo systems. Expression of a construct containing ORF8a and ORF8b generated only a single protein, 8a; no 8b protein expression was obtained. Expression of a construct containing ORF8 generated the 8ab fusion protein. Site‐directed mutagenesis and enzymatic treatment revealed that protein 8ab is modified by N‐linked glycosylation on the N81 residue and by ubiquitination. In the absence of the 8a region, protein 8b undergoes rapid degradation by proteasomes, and addition of proteasome inhibitors inhibits the degradation of protein 8b as well as the protein 8b‐induced rapid degradation of the severe acute respiratory syndrome coronavirus E protein. Glycosylation could also stabilize protein 8ab. More interestingly, the two proteins could bind to monoubiquitin and polyubiquitin, suggesting the potential involvement of these proteins in the pathogenesis of severe acute respiratory syndrome coronavirus.

Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. The other human coronaviruses (HCoV)-HCoV-229E, HCoV-OC43, NL63, and HKU1-are collectively responsible for about 10 to 30% of common colds. Generally harmless and selflimiting, these HCoV are also implicated in severe clinical outcomes, particularly in immunocompromised individuals, infants, and the elderly (3). Other coronaviruses cause considerable economic concern to the livestock industry as they readily infect farmed animals such as cows (4), pigs (5), and chickens (6).In addition to the diverse range of species that they infect, coronaviruses have a propensity for host switching. For instance, HCoV-OC43 bears a strong resemblance to a bovine coronavirus, from which it probably originated (7). SARS-CoV is postulated to have originated from bats and then transferred to palm civets and finally humans (8). The MERS coronavirus probably also has its origin in bats and is responsible for severe respiratory and renal failure in humans (9). Although human-to-human transmission is low at present (10), this new beta-coronavirus has raised global health concerns because its mortality rate is more than 30% (1, 11). This "interspecies jumping" continuously threatens to initiate a novel epidemic and presents a challenge for vaccine-based containment.It is thus critical to have a better understanding of the infectious cycle of CoV. This multistep process includes attachment of the spike protein (S) to cell surface receptors, endocytosis, and then fusion of the viral and endocytic membranes (12). The viral capsid then undergoes an uncoating process to deliver the viral genome into the cytosol.Host ribosomes then translate the viral genome, yielding nonstructural proteins that modulate virus pathogenesis (13) and form with host membranes the viral transcription/replication complex (RTC). The RTC is responsible for transcription of full-length genomic RNA as well as subgenomic RNA species via a nidovirusspecific discontinuous tran...

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Type I collagen is a key protein of most connective tissue and its up-regulation is required for wound healing but is also involved in fibrosis. Control of expression of this collagen remains poorly understood apart from Transforming Growth Factor beta (TGF-β1)-mediated induction. To generate a sensitive, practical, robust, image-based high-throughput-compatible reporter system, we genetically inserted a short-lived fluorescence reporter downstream of the endogenous type I collagen (COL1A1) promoter in skin fibroblasts. Using a variety of controls, we demonstrate that the cell line faithfully reports changes in type I collagen expression with at least threefold enhanced sensitivity compared to endogenous collagen monitoring. We use this assay to test the potency of anti-fibrotic compounds and screen siRNAs for regulators of TGF-β1-induced type I collagen expression. We propose our reporter cell line, Red-COLA1, as a new efficient tool to study type I collagen transcriptional regulation.

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