Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells.

Coronavirus tropism is predominantly determined by the interaction between coronavirus spikes and the host receptors. In this regard, coronaviruses have evolved a complicated receptor-recognition system through their spike proteins. Spikes from highly related coronaviruses can recognize distinct receptors, whereas spikes of distant coronaviruses can employ the same cell-surface molecule for entry. Moreover, coronavirus spikes can recognize a broad range of cell-surface molecules in addition to the receptors and thereby can augment coronavirus attachment or entry. The receptor of Middle East respiratory syndrome coronavirus (MERS-CoV) is dipeptidyl peptidase 4 (DPP4). In this study, we identified membrane-associated 78-kDa glucose-regulated protein (GRP78) as an additional binding target of the MERS-CoV spike. Further analyses indicated that GRP78 could not independently render nonpermissive cells susceptible to MERS-CoV infection but could facilitate MERS-CoV entry into permissive cells by augmenting virus attachment. More importantly, by exploring potential interactions between GRP78 and spikes of other coronaviruses, we discovered that the highly conserved human GRP78 could interact with the spike protein of bat coronavirus HKU9 (bCoV-HKU9) and facilitate its attachment to the host cell surface. Taken together, our study has identified GRP78 as a host factor that can interact with the spike proteins of two Betacoronaviruses, the lineage C MERS-CoV and the lineage D bCoV-HKU9. The capacity of GRP78 to facilitate surface attachment of both a human coronavirus and a phylogenetically related bat coronavirus exemplifies the need for continuous surveillance of the evolution of animal coronaviruses to monitor their potential for human adaptations.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 Is an Important Surface Attachment Factor That Facilitates Entry of Middle East Respiratory Syndrome Coronavirus.

UNLABELLED: The spike proteins of coronaviruses are capable of binding to a wide range of cellular targets, which contributes to the broad species tropism of coronaviruses. Previous reports have demonstrated that Middle East respiratory syndrome coronavirus (MERS-CoV) predominantly utilizes dipeptidyl peptidase 4 (DPP4) for cell entry. However, additional cellular binding targets of the MERS-CoV spike protein that may augment MERS-CoV infection have not been further explored. In the current study, using the virus overlay protein binding assay (VOPBA), we identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV. CEACAM5 coimmunoprecipitated with the spike protein of MERS-CoV in both overexpressed and endogenous settings. Disrupting the interaction between CEACAM5 and MERS-CoV spike with anti-CEACAM5 antibody, recombinant CEACAM5 protein, or small interfering RNA (siRNA) knockdown of CEACAM5 significantly inhibited the entry of MERS-CoV. Recombinant expression of CEACAM5 did not render nonpermissive baby hamster kidney (BHK21) cells susceptible to MERS-CoV infection. Instead, CEACAM5 overexpression significantly enhanced the attachment of MERS-CoV to the BHK21 cells. More importantly, the entry of MERS-CoV was increased when CEACAM5 was overexpressed in permissive cells, which suggested that CEACAM5 could facilitate MERS-CoV entry in conjunction with DPP4 despite not being able to support MERS-CoV entry independently. Taken together, the results of our study identified CEACAM5 as a novel cell surface binding target of MERS-CoV that facilitates MERS-CoV infection by augmenting the attachment of the virus to the host cell surface. IMPORTANCE: Infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with the highest mortality rate among all known human-pathogenic coronaviruses. Currently, there are no approved vaccines or therapeutics against MERS-CoV infection. The identification of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a novel cell surface binding target of MERS-CoV advanced our knowledge on the cell binding biology of MERS-CoV. Importantly, CEACAM5 could potentiate the entry of MERS-CoV by functioning as an attachment factor. In this regard, CEACAM5 could serve as a novel target, in addition to dipeptidyl peptidase-4 (DPP4), in the development of antiviral strategies for MERS-CoV.

Hemagglutinin of influenza A virus binds specifically to cell surface nucleolin and plays a role in virus internalization.

The hemagglutinin (HA) protein of influenza A virus initiates cell entry by binding to sialic acids on target cells. In the current study, we demonstrated that in addition to sialic acids, influenza A/Puerto Rico/8/34 H1N1 (PR8) virus HA specifically binds to cell surface nucleolin (NCL). The interaction between HA and NCL was initially revealed with virus overlay protein binding assay (VOPBA) and subsequently verified with co-immunoprecipitation. Importantly, inhibiting cell surface NCL with NCL antibody, blocking PR8 viruses with purified NCL protein, or depleting endogenous NCL with siRNA all substantially reduced influenza virus internalization. We further demonstrated that NCL was a conserved cellular factor required for the entry of multiple influenza A viruses, including H1N1, H3N2, H5N1, and H7N9. Overall, our findings identified a novel role of NCL in influenza virus life cycle and established NCL as one of the host cell surface proteins for the entry of influenza A virus.

Paralogous metabolism: S-alkyl-cysteine degradation in Bacillus subtilis.

Metabolism is prone to produce analogs of essential building blocks in the cell (here named paralogous metabolism). The variants result from lack of absolute accuracy in enzyme-templated reactions as well as from molecular aging. If variants were left to accumulate, the earth would be covered by chemical waste. The way bacteria cope with this situation is essentially unexplored. To gain a comprehensive understanding of Bacillus subtilis sulphur paralogous metabolism, we used expression profiling with DNA arrays to investigate the changes in gene expression in the presence of S-methyl-cysteine (SMeC) and its close analog, methionine, as sole sulphur source. Altogether, more than 200 genes whose relative strength of induction was significantly different depending on the sulphur source used were identified. This allowed us to pinpoint operon ytmItcyJKLMNytmO_ytnIJ_rbfK_ytnLM as controlling the pathway cycling SMeC directly to cysteine, without requiring sulphur oxygenation. Combining genetic and physiological experiments, we deciphered the corresponding pathway that begins with protection of the metabolite by acetylation. Oxygenation of the methyl group then follows, and after deprotection (deacetylation), N-formyl cysteine is produced. This molecule is deformylated by the second deformylase present in B. subtilis DefB, yielding cysteine. This pathway appears to be present in plant-associated microbes.

Unraveling the molecular basis of temperature-dependent genetic regulation in Penicillium marneffei.

Penicillium marneffei is an opportunistic fungal pathogen endemic in Southeast Asia, causing lethal systemic infections in immunocompromised patients. P. marneffei grows in a mycelial form at the ambient temperature of 25°C and transitions to a yeast form at 37°C. The ability to alternate between the mycelial and yeast forms at different temperatures, namely, thermal dimorphism, has long been considered critical for the pathogenicity of P. marneffei, yet the underlying genetic mechanisms remain elusive. Here we employed high-throughput sequencing to unravel global transcriptional profiles of P. marneffei PM1 grown at 25 and 37°C. Among ∼11,000 protein-coding genes, 1,447 were overexpressed and 1,414 were underexpressed at 37°C. Counterintuitively, heat-responsive genes, predicted in P. marneffei through sequence comparison, did not tend to be overexpressed at 37°C. These results suggest that P. marneffei may take a distinct strategy of genetic regulation at the elevated temperature; the current knowledge concerning fungal heat response, based on studies of model fungal organisms, may not be applicable to P. marneffei. Our results further showed that the tandem repeat sequences (TRSs) are overrepresented in coding regions of P. marneffei genes, and TRS-containing genes tend to be overexpressed at 37°C. Furthermore, genomic sequences and expression data were integrated to characterize gene clusters, multigene families, and species-specific genes of P. marneffei. In sum, we present an integrated analysis and a comprehensive resource toward a better understanding of temperature-dependent genetic regulation in P. marneffei.

Immunoassays based on Penicillium marneffei Mp1p derived from Pichia pastoris expression system for diagnosis of penicilliosis.

BACKGROUND: Penicillium marneffei is a dimorphic fungus endemic in Southeast Asia. It can cause fatal penicilliosis in humans, particularly in HIV-infected people. Diagnosis of this infection is difficult because its clinical manifestations are not distinctive. Specialized laboratory tests are necessary to establish a definitive diagnosis for successful management. We have demonstrated previously that a cell wall mannoprotein Mp1p, abundant in P. marneffei, is a potential biomarker for diagnosis of P. marneffei infections. In the present study, we describe immunoassays based on Mp1p derived from the yeast Pichia pastoris expression system. METHODOLOGY/PRINCIPAL FINDINGS: We generated monoclonal antibodies (MAbs) and rabbit polyclonal antibodies (PAbs) against Mp1p expressed in P. pastoris. Subsequently, we developed two Mp1p antigen capture ELISAs which employed MAbs for both the capture and detecting antibodies (MAb-MAb pair) or PAbs and MAbs as the capture and detecting antibodies (PAbs-MAb pair) respectively. The two Mp1p antigen ELISAs detected Mp1p specifically in cultures of P. marneffei yeast phase at 37-40°C and had no cross-reaction with other tested pathogenic fungi. The sensitivities and specificities of the two antigen assays were found to be 55% (11/20) and 99.6% (538/540) for MAb-MAb Mp1p ELISA, and 75% (15/20) and 99.4% (537/540) for PAbs-MAb Mp1p ELISA performed using 20 sera with culture-confirmed penicilliosis, and 540 control sera from 15 other mycosis patients and 525 healthy donors. Meanwhile, we also developed an anti-Mp1p IgG antibody ELISA with an evaluated sensitivity of 30% (6/20) and a specificity of 98.5% (532/540) using the same sera. Furthermore, combining the results of Mp1p antigen and antibody detection improved the sensitivity of diagnosis to 100% (20/20). CONCLUSIONS/SIGNIFICANCE: Simultaneous detection of antigen and antibody using the immunoassays based on Mp1p derived from P. pastoris greatly improves detection sensitivity. The procedures should be useful for the routine diagnosis of penicilliosis.

Draft genome sequence of Penicillium marneffei strain PM1.

Penicillium marneffei is the most important thermal dimorphic, pathogenic fungus endemic in China and Southeast Asia and is particularly important in HIV-positive patients. We report the 28,887,485-bp draft genome sequence of P. marneffei, which contains its complete mitochondrial genome, sexual cycle genes, a high diversity of Mp1p homologues, and polyketide synthase genes.

Identification of influenza A nucleoprotein as an antiviral target.

Influenza A remains a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. Antiviral resistance to available drugs such as adamantanes or neuraminidase inhibitors has appeared rapidly, creating a need for new antiviral targets and new drugs for influenza virus infections. Using forward chemical genetics, we have identified influenza A nucleoprotein (NP) as a druggable target and found a small-molecule compound, nucleozin, that triggers the aggregation of NP and inhibits its nuclear accumulation. Nucleozin impeded influenza A virus replication in vitro with a nanomolar median effective concentration (EC(50)) and protected mice challenged with lethal doses of avian influenza A H5N1. Our results demonstrate that viral NP is a valid target for the development of small-molecule therapies.

Spike protein, S, of human coronavirus HKU1: role in viral life cycle and application in antibody detection.

We recently described the discovery, genome, clinical features, genotypes and evolution of a novel and global human respiratory virus named human coronavirus HKU1 (HCoV-HKU1) which is not yet culturable. We expressed a C-terminal FLAG-tagged CoV-HKU1 spike (S) protein by the Semliki Forest Virus (SFV) system and investigated its maturation profile. Pulse chase labeling revealed that S-FLAG was expressed as high-mannose N-glycans of monomers and trimers. It was predominantly cleaved into subdomains S1 and S2 during maturation. S1 was secreted into the medium. Immunofluorescence analysis visualized S along the secretory pathway from endoplasmic reticulum to plasma membrane. Cleavage of S and release of HCoV-HKU1 S pseudotyped virus were inhibited by furin or furin-like enzyme inhibitors. The cell-based expressed full-length S-FLAG could be recognized by the convalescent serum obtained from a patient with HCoV-HKU1 pneumonia. The data suggest that the native form of HCoV-HKU1 spike expressed in our system can be used in developing serological diagnostic assay and in understanding the role of S in the viral life cycle.

Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E.

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.

Detection of cell wall galactomannoprotein Afmp1p in culture supernatants of Aspergillus fumigatus and in sera of aspergillosis patients.

Mannoproteins are important and abundant structural components of fungal cell walls. The AFMP1 gene encodes a cell wall galactomannoprotein of Aspergillus fumigatus. In the present study, we show that Afmp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Afmp1p was capable of detecting this protein from the cell culture supernatant of A. fumigatus. The anti-Afmp1p antibody is specific since it fails to react with any protein from lysates of Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Penicillium marneffei, Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum by Western blotting. In addition, this Afmp1p antigen-based ELISA is also specific for A. fumigatus since the cell culture supernatants of the other eight fungi gave negative results. Finally, a clinical evaluation of sera from invasive aspergillosis patients indicates that 8 of 15 (53%) patients are Afmp1p antigen test positive. Furthermore, an Afmp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for invasive aspergillosis carry a sensitivity of 86.7% (13 of 15). The specificities of the tests are high since none of the 138 control sera, including 100 from normal blood donors, 20 from patients with penicilliosis marneffei, 6 from patients with candidemia, 8 from patients with typhoid fever, and 4 from patients with melioidosis, was positive by either test. In conclusion, the combined Afmp1p antibody and antigen tests are highly sensitive and specific for A. fumigatus invasive aspergillosis.

Detection of antibodies specific to an antigenic cell wall galactomannoprotein for serodiagnosis of Aspergillus fumigatus aspergillosis.

Aspergilloma and invasive aspergillosis are important opportunistic infections caused by Aspergillus species, among which Aspergillus fumigatus is the most common species associated with human disease. We developed an enzyme-linked immunosorbent assay (ELISA)-based antibody assay with Afmp1p, a purified recombinant antigenic cell wall galactomannoprotein of A. fumigatus. Evaluation of the test with guinea pig sera against A. fumigatus and other pathogenic fungi indicated that this assay was specific for A. fumigatus. Clinical evaluation revealed that the assay was 100% sensitive for patients with aspergilloma and 33.3% sensitive for patients with invasive aspergillosis. No false-positive results were found for serum samples from 80 healthy blood donors, 6 patients with typhoid fever, 4 patients with melioidosis, 20 patients with penicilliosis marneffei, 5 patients with candidiasis, and 4 patients with cryptococcosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Afmp1p antibody can be of significant value as a diagnostic for aspergillosis.