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  • 1
    Publication Date: 2018-07-18
    Description: Coronavirus replication is closely associated with the endoplasmic reticulum (ER), the primary cellular organelle for protein synthesis, folding, and modification. ER stress is a common consequence in coronavirus-infected cells. However, how the virus-induced ER stress influences coronavirus replication and pathogenesis remains controversial. Here, we demonstrated that infection with the alphacoronavirus transmissible gastroenteritis virus (TGEV) induced ER stress and triggered the unfolded protein response (UPR) in vitro and in vivo , and ER stress negatively regulated TGEV replication in vitro . Although TGEV infection activated all three UPR pathways (activating transcription factor 6 [ATF6], inositol-requiring enzyme 1 [IRE1], and protein kinase R-like ER kinase [PERK]), the virus-triggered UPR suppressed TGEV replication in both swine testicular (ST) and IPEC-J2 cells primarily through activation of the PERK-eukaryotic initiation factor 2α (eIF2α) axis, as shown by functional studies with overexpression, small interfering RNA (siRNA), or specific chemical inhibitors. Moreover, we demonstrated that PERK-eIF2α axis-mediated inhibition of TGEV replication occurs through phosphorylated eIF2α-induced overall attenuation of protein translation. In addition to direct inhibition of viral production, the PERK-eIF2α pathway activated NF-B and then facilitated type I IFN production, resulting in TGEV suppression. Taken together, our results suggest that the TGEV-triggered PERK-eIF2α pathway negatively regulates TGEV replication and represents a vital aspect of host innate responses to invading pathogens. IMPORTANCE The induction of ER stress is a common outcome in cells infected with coronaviruses. The UPR initiated by ER stress is actively involved in viral replication and modulates the host innate responses to the invading viruses, but these underlying mechanisms remain incompletely understood. We show here that infection with the alphacoronavirus TGEV elicited ER stress in vitro and in vivo , and the UPR PERK-eIF2α branch was predominantly responsible for the suppression of TGEV replication by ER stress. Furthermore, the PERK-eIF2α axis inhibited TGEV replication through direct inhibition of viral proteins due to global translation inhibition and type I IFN induction. These findings highlight a critical role of the UPR PERK-eIF2α pathway in modulating host innate immunity and coronavirus replication.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
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  • 2
    Publication Date: 2018-05-15
    Description: The primate lentiviral accessory protein Nef downregulates CD4 and major histocompatibility complex class I (MHC-I) from the cell surface via independent endosomal trafficking pathways to promote viral pathogenesis. In addition, Nef antagonizes a novel restriction factor, SERINC5 (Ser5), to increase viral infectivity. To explore the molecular mechanism of Ser5 antagonism by Nef, we determined how Nef affects Ser5 expression and intracellular trafficking in comparison to CD4 and MHC-I. We confirm that Nef excludes Ser5 from human immunodeficiency virus type 1 (HIV-1) virions by downregulating its cell surface expression via similar functional motifs required for CD4 downregulation. We find that Nef decreases both Ser5 and CD4 expression at steady-state levels, which are rescued by NH 4 Cl or bafilomycin A1 treatment. Nef binding to Ser5 was detected in living cells using a bimolecular fluorescence complementation assay, where Nef membrane association is required for interaction. In addition, Nef triggers rapid Ser5 internalization via receptor-mediated endocytosis and relocalizes Ser5 to Rab5 + early, Rab7 + late, and Rab11 + recycling endosomes. Manipulation of AP-2, Rab5, Rab7, and Rab11 expression levels affects the Nef-dependent Ser5 and CD4 downregulation. Moreover, although Nef does not promote Ser5 polyubiquitination, Ser5 downregulation relies on the ubiquitination pathway, and both K48- and K63-specific ubiquitin linkages are required for the downregulation. Finally, Nef promotes Ser5 colocalization with LAMP1, which is enhanced by bafilomycin A1 treatment, suggesting that Ser5 is targeted to lysosomes for destruction. We conclude that Nef uses a similar mechanism to downregulate Ser5 and CD4, which sorts Ser5 into a point-of-no-return degradative pathway to counteract its restriction. IMPORTANCE Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) express an accessory protein called Nef to promote viral pathogenesis. Nef drives immune escape in vivo through downregulation of CD4 and MHC-I from the host cell surface. Recently, Nef was reported to counteract a novel host restriction factor, Ser5, to increase viral infectivity. Nef downregulates cell surface Ser5, thus preventing its incorporation into virus particles, resulting in disruption of its antiviral activity. Here, we report mechanistic studies of Nef-mediated Ser5 downregulation in comparison to CD4 and MHC-I. We demonstrate that Nef binds directly to Ser5 in living cells and that Nef-Ser5 interaction requires Nef association with the plasma membrane. Subsequently, Nef internalizes Ser5 from the plasma membrane via receptor-mediated endocytosis, and targets ubiquitinated Ser5 to endosomes and lysosomes for destruction. Collectively, these results provide new insights into our ongoing understanding of the Nef-Ser5 arms race in HIV-1 infection.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
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  • 3
    Publication Date: 2018-03-23
    Description: Rats vary in their susceptibilities to Toxoplasma gondii infection depending on the rat strain. Compared to the T. gondii -susceptible Brown Norway (BN) rat, the Lewis (LEW) rat is extremely resistant to T. gondii . Thus, these two rat strains are ideal models for elucidating host mechanisms that are important for host resistance to T. gondii infection. Therefore, in our efforts to unravel molecular factors directing the protective early innate immune response in the LEW rat, we performed RNA sequencing analysis of the LEW versus BN rat with or without T. gondii infection. We identified three candidate small G TPase im munity- a ssociated p roteins (GIMAPs) that were upregulated (false discovery rate, 0.05) in the LEW rat in response to T. gondii infection. Subsequently, we engineered T. gondii -susceptible NR8383 rat macrophage cells for overexpression of LEW rat-derived candidate GIMAP 4, 5, and 6. By immunofluorescence analysis we observed that GIMAP 4, 5, and 6 in T. gondii -infected NR8383 cells each colocalized with GRA5, a parasite parasitophorous vacuole membrane (PVM) marker protein, suggesting their translocation to the PVM. Interestingly, overexpression of each candidate GIMAP in T. gondii -infected NR8383 cells induced translocation of LAMP1, a lysosome marker protein, to the T. gondii surface membrane. Importantly, overexpression of GIMAP 4, 5, or 6 individually inhibited intracellular T. gondii growth, with GIMAP 4 having the highest inhibitory effect. Together, our findings indicate that upregulation of GIMAP 4, 5, and 6 contributes to the robust refractoriness of the LEW rat to T. gondii through induction of lysosomal fusion to the otherwise nonfusogenic PVM.
    Print ISSN: 0019-9567
    Electronic ISSN: 1098-5522
    Topics: Medicine
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  • 4
    Publication Date: 2018-03-28
    Description: A novel 65.8-kb multidrug resistance transposon, designated Tn 6450 , was characterized in a Proteus mirabilis isolate from chicken in China. Tn 6450 contains 18 different antimicrobial resistance genes, including cephalosporinase gene bla DHA-1 and fluoroquinolone resistance genes qnrA1 and aac(6 ' )-Ib-cr . It carries a class 1/2 hybrid integron composed of intI2 and a 3' conserved segment of the class 1 integron. Tn 6450 is derived from Tn 7 via acquisition of new mobile elements and resistance genes.
    Print ISSN: 0066-4804
    Electronic ISSN: 1098-6596
    Topics: Biology , Medicine
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  • 5
    Publication Date: 2018-04-27
    Description: Through some specific amino acid residues, cofilin, a ubiquitous actin depolymerization factor, can significantly affect mitochondrial function related to drug resistance and apoptosis in Saccharomyces cerevisiae ; however, this modulation in a major fungal pathogen, Aspergillus fumigatus , was still unclear. Hereby, it was found, first, that mutations on several charged residues in cofilin to alanine, D19A-R21A, E48A, and K36A, increased the formation of reactive oxygen species and induced apoptosis along with typical hallmarks, including mitochondrial membrane potential depolarization, cytochrome c release, upregulation of metacaspases, and DNA cleavage, in A. fumigatus . Two of these mutations (D19A-R21A and K36A) increased acetyl coenzyme A and ATP concentrations by triggering fatty acid β-oxidation. The upregulated acetyl coenzyme A affected the ergosterol biosynthetic pathway, leading to overexpression of cyp51A and - B , while excess ATP fueled ATP-binding cassette transporters. Besides, both of these mutations reduced the susceptibility of A. fumigatus to azole drugs and enhanced the virulence of A. fumigatus in a Galleria mellonella infection model. Taken together, novel and key charged residues in cofilin were identified to be essential modules regulating the mitochondrial function involved in azole susceptibility, apoptosis, and virulence of A. fumigatus .
    Print ISSN: 0066-4804
    Electronic ISSN: 1098-6596
    Topics: Biology , Medicine
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  • 6
    Publication Date: 2018-03-29
    Description: The group of highly related avian leukosis viruses (ALVs) in chickens are thought to have evolved from a common retroviral ancestor into six subgroups, A to E and J. These ALV subgroups use diverse cellular proteins encoded by four genetic loci in chickens as receptors to gain entry into host cells. Hosts exposed to ALVs might be under selective pressure to develop resistance to ALV infection. Indeed, resistance alleles have previously been identified in all four receptor loci in chickens. The tvb gene encodes a receptor, which determines the susceptibility of host cells to ALV subgroup B (ALV-B), ALV-D, and ALV-E. Here we describe the identification of two novel alleles of the tvb receptor gene, which possess independent insertions each within exon 4. The insertions resulted in frameshift mutations that reveal a premature stop codon that causes nonsense-mediated decay of the mutant mRNA and the production of truncated Tvb protein. As a result, we observed that the frameshift mutations in the tvb gene significantly lower the binding affinity of the truncated Tvb receptors for the ALV-B, ALV-D, and ALV-E envelope glycoproteins and significantly reduce susceptibility to infection by ALV-B, ALV-D and ALV-E in vitro and in vivo . Taken together, these findings suggest that frameshift mutation can be a molecular mechanism of reducing susceptibility to ALV and enhance our understanding of virus-host coevolution. IMPORTANCE Avian leukosis virus (ALV) once caused devastating economic loss to the U.S. poultry industry prior the current eradication schemes in place, and it continues to cause severe calamity to the poultry industry in China and Southeast Asia, where deployment of a complete eradication scheme remains a challenge. The tvb gene encodes the cellular receptor necessary for subgroup B, D, and E ALV infection. Two tvb allelic variants that resulted from frameshift mutations have been identified in this study, which have been shown to have significantly reduced functionality in mediating subgroup B, D, and E ALV infection. Unlike the control of herpesvirus-induced diseases by vaccination, the control of avian leukosis in chickens has relied totally on virus eradication measures and host genetic resistance. This finding enriches the allelic pool of the tvb gene and expands the potential for genetic improvement of ALV resistance in varied chicken populations by selection.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
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  • 7
    Publication Date: 2018-01-03
    Description: Coxsackievirus A6 (CVA6) has recently emerged as one of the predominant causative agents of hand, foot, and mouth disease (HFMD). The structure of the CVA6 mature viral particle has not been solved thus far. Our previous work shows that recombinant virus-like particles (VLPs) of CVA6 represent a promising CVA6 vaccine candidate. Here, we report the first cryo-electron microscopy (cryo-EM) structure of the CVA6 VLP at 3.0-Å resolution. The CVA6 VLP exhibits the characteristic features of enteroviruses but presents an open channel at the 2-fold axis and an empty, collapsed VP1 pocket, which is broadly similar to the structures of the enterovirus 71 (EV71) VLP and coxsackievirus A16 (CVA16) 135S expanded particle, indicating that the CVA6 VLP is in an expanded conformation. Structural comparisons reveal that two common salt bridges within protomers are maintained in the CVA6 VLP and other viruses of the Enterovirus genus, implying that these salt bridges may play a critical role in enteroviral protomer assembly. However, there are apparent structural differences among the CVA6 VLP, EV71 VLP, and CVA16 135S particle in the surface-exposed loops and C termini of subunit proteins, which are often antigenic sites for enteroviruses. By immunological assays, we identified two CVA6-specific linear B-cell epitopes (designated P42 and P59) located at the GH loop and the C-terminal region of VP1, respectively, in agreement with the structure-based prediction of antigenic sites. Our findings elucidate the structural basis and important antigenic sites of the CVA6 VLP as a strong vaccine candidate and also provide insight into enteroviral protomer assembly. IMPORTANCE Coxsackievirus A6 (CVA6) is becoming one of the major pathogens causing hand, foot, and mouth disease (HFMD), leading to significant morbidity and mortality in children and adults. However, no vaccine is currently available to prevent CVA6 infection. Our previous work shows that recombinant virus-like particles (VLPs) of CVA6 are a promising CVA6 vaccine candidate. Here, we present a 3.0-Å structure of the CVA6 VLP determined by cryo-electron microscopy. The overall architecture of the CVA6 VLP is similar to those of the expanded structures of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), but careful structural comparisons reveal significant differences in the surface-exposed loops and C termini of each capsid protein of these particles. In addition, we identified two CVA6-specific linear B-cell epitopes and mapped them to the GH loop and the C-terminal region of VP1, respectively. Collectively, our findings provide a structural basis and important antigenic information for CVA6 VLP vaccine development.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
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