人星状病毒研究进展

人星状病毒是引起婴幼儿病毒性腹泻的重要病原之一,本文回顾了其分子生物学特征、型别、所致疾病特征、致病机制、流行病学特征及检测方法等方面的文献,指出人星状病毒目前存在多种型别,尤其是近年来发现了多种新型的星状病毒,主要的流行型别是血清型1型,不仅引起肠道内感染,还可以引起其他系统疾病,所致疾病特征和其他腹泻相关病毒相似,但是其致病机制的研究尚不清楚,对于几种新发现的星状病毒,其与疾病的相关性研究较少,且未建立完善的实验室检测方法,还需要进一步的研究。     

Novel Insights into Cell Entry of Emerging Human Pathogenic Arenaviruses

Viral hemorrhagic fevers caused by emerging RNA viruses of the Arenavirus family are among the most devastating human diseases. Climate change, global trade, and increasing urbanization promote the emergence and re-emergence of these human pathogenic viruses. Emerging pathogenic arenaviruses are of zoonotic origin and reservoir-to-human transmission is crucial for spillover into human populations. Host cell attachment and entry are the first and most fundamental steps of every virus infection and represent major barriers for zoonotic transmission. During host cell invasion, viruses critically depend on cellular factors, including receptors, co-receptors, and regulatory proteins of endocytosis. An in-depth understanding of the complex interaction of a virus with cellular factors implicated in host cell entry is therefore crucial to predict the risk of zoonotic transmission, define the tissue tropism, and assess disease potential. Over the past years, investigation of the molecular and cellular mechanisms underlying host cell invasion of human pathogenic arenaviruses uncovered remarkable viral strategies and provided novel insights into viral adaptation and virus–host co-evolution that will be covered in the present review.     

Processing of Nonstructural Protein 1a of Human Astrovirus

Astrovirus contains three open reading frames (ORF) on its genomic RNA, ORF1a, ORF1b, and ORF2. ORF1a encodes a 920-amino-acid (aa) nonstructural protein, nsP1a, which displays a 3C-like serine protease motif. Little is known about the processing of nsP1a or whether the protease it contains is active and involved in autocatalytic processing. Here we address both of these matters. Intact and N-terminally deleted forms of ORF1a from human astrovirus serotype 1 were expressed in BHK cells, and nsP1a-derived processing products were immunoprecipitated with an nsP1a-specific antibody or an antibody specific for an N-terminally linked epitope tag. The mapping of the main processing products, p20 and p27, suggests cleavage sites near aa 170, 410, and 655 of nsP1a. Cleavages at around aa 410 and 655, but not aa 170, were abolished when a 9-aa substitution was introduced into the protease motif in nsP1a. The p27 processing product was also found in Caco-2 cells that had been infected with human astrovirus serotype 1, confirming the presence of the cleavage sites at approximately aa 410 and 655.     

Isolation and Characterization of a Near-Haploid Human Cell Line

Mammalian somatic cells are usually diploid. Occasional rare human tumors have been shown to have a hypodiploid karyotype. We have isolated a near-haploid subclone (P1-55) from a heterogeneous human leukemia cell line, KBM-7. These near-haploid cells have approximately half the human diploid DNA content and have a haploid karyotype except for a disomy of chromosome 8 (25, XY, +8, Ph(+)). This cell line maintains a majority of cells with a near-haploid karyotype for at least 12 weeks in culture. By serial subcloning, we have isolated near-haploid subclones that maintain ploidy for at least 8 months in culture. Near-haploid cells can also be efficiently isolated from mixed ploidy cultures by size selection. The availability of this human near-haploid cell line should facilitate the genetic analysis of cultured human cells.     

Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin

The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin α_Vβ₃ blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. α_Vβ₃ blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving α_Vβ₃. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.     

Type I Interferon Response Is Delayed in Human Astrovirus Infections

Type I interferon (IFN) activation and its subsequent effects are important in the response to viral infections. Here we show that human astroviruses (HAstVs), which are important agents of acute gastroenteritis in children, induce a mild and delayed IFN response upon infecting CaCo-2 cells. Although IFN-β mRNA is detected within infected cells and supernatant from infected cells show antiviral activity against the replication of other well-known IFN-sensitive viruses, these responses occur at late stages of infection once genome replication has taken place. On the other hand, HAstV replication can be partially reduced by the addition of exogenous IFN, and inhibition of IFN activation by BX795 enhances viral replication, indicating that HAstVs are IFN-sensitive viruses. Finally, different levels of IFN response were observed in cells infected with different HAstV mutants with changes in the hypervariable region of nsP1a/4, suggesting that nsP1a/4 genotype may potentially have clinical implications due to its correlation with the viral replication phenotype and the antiviral responses induced within infected cells.     

Antigenic Characterization of the HCMV gH/gL/gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies

Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV.     

Identification of TRAPPC8 as a Host Factor Required for Human Papillomavirus Cell Entry

Human papillomavirus (HPV) is a non-enveloped virus composed of a circular DNA genome and two capsid proteins, L1 and L2. Multiple interactions between its capsid proteins and host cellular proteins are required for infectious HPV entry, including cell attachment and internalization, intracellular trafficking and viral genome transfer into the nucleus. Using two variants of HPV type 51, the Ma and Nu strains, we have previously reported that MaL2 is required for efficient pseudovirus (PsV) transduction. However, the cellular factors that confer this L2 dependency have not yet been identified. Here we report that the transport protein particle complex subunit 8 (TRAPPC8) specifically interacts with MaL2. TRAPPC8 knockdown in HeLa cells yielded reduced levels of reporter gene expression when inoculated with HPV51Ma, HPV16, and HPV31 PsVs. TRAPPC8 knockdown in HaCaT cells also showed reduced susceptibility to infection with authentic HPV31 virions, indicating that TRAPPC8 plays a crucial role in native HPV infection. Immunofluorescence microscopy revealed that the central region of TRAPPC8 was exposed on the cell surface and colocalized with inoculated PsVs. The entry of Ma, Nu, and L2-lacking PsVs into cells was equally impaired in TRAPPC8 knockdown HeLa cells, suggesting that TRAPPC8-dependent endocytosis plays an important role in HPV entry that is independent of L2 interaction. Finally, expression of GFP-fused L2 that can also interact with TRAPPC8 induced dispersal of the Golgi stack structure in HeLa cells, a phenotype also observed by TRAPPC8 knockdown. These results suggest that during viral intracellular trafficking, binding of L2 to TRAPPC8 inhibits its function resulting in Golgi destabilization, a process that may assist HPV genome escape from the trans-Golgi network.     

Characterization of Cell Surface Lectin-binding Patterns of Human Airway Epithelium

Glycosylated structures on the cell surface have a role in cell adhesion, migration, and proliferation. Repair of the airway epithelium after injury requires each of these processes, but the normal cell surface glycosylation of non-mucin producing airway epithelial cells is unknown. We examined cell surface glycosylation in human airway epithelial cells in tissue sections and in human airway epithelial cell lines in culture. Thirty-eight lectin probes were used to determine specific carbohydrate residues by lectin-histochemistry. Galactose or galactosamine-specific lectins labeled basal epithelial cells, lectins specific for several different carbohydrate structures bound columnar epithelial cells, and fucose-specific lectins labeled all airway epithelial cells. The epithelial cell lines 1HAEo– and 16HBE14o– bound lectins that were specific to basal epithelial cells. Flow cytometry of these cell lines with selected lectins demonstrated that lectin binding was to cell surface carbohydrates, and revealed possible hidden tissue antigens on dispersed cultured cells. We demonstrate specific lectin-binding patterns on the surface of normal human airway epithelial cells. The expression of specific carbohydrate residues may be useful to type epithelial cells and as a tool to examine cell events involved in epithelial repair.     

Characterization of Ebola Virus Entry by Using Pseudotyped Viruses: Identification of Receptor-Deficient Cell Lines

Studies analyzing Ebola virus replication have been severely hampered by the extreme pathogenicity of this virus. To permit analysis of the host range and function of the Ebola virus glycoprotein (Ebo-GP), we have developed a system for pseudotyping these glycoproteins into murine leukemia virus (MLV). This pseudotyped virus, MLV(Ebola), can be readily concentrated to titers which exceed 5 × 10⁶ infectious units/ml and is effectively neutralized by antibodies specific for Ebo-GP. Analysis of MLV(Ebola) infection revealed that the host range conferred by Ebo-GP is very broad, extending to cells of a variety of species. Notably, all lymphoid cell lines tested were completely resistant to infection; we speculate that this is due to the absence of a cellular receptor for Ebo-GP on B and T cells. The generation of high-titer MLV(Ebola) pseudotypes will be useful for the analysis of immune responses to Ebola virus infection, development of neutralizing antibodies, analysis of glycoprotein function, and isolation of the cellular receptor(s) for the Ebola virus.     

Structural Correlates of Rotavirus Cell Entry

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex—for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼700 Å inner capsid particle (“double-layered particle”, DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.     

Pharmacological Characterization of Cholinergic Receptors in a Human Neuroblastoma Cell Line

A human neuroblastoma cell line, IMR32, has been characterized as far as morphology, membrane receptors for neurotransmitters, and uptake and release of [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine). These cells expressed at their surface both nicotinic and muscarinic cholinergic receptors, revealed by [125I]alpha-bungarotoxin and [3H]quinuclidinylbenzilate ([3H]QNB) binding, respectively. [125I]alpha-Bungarotoxin binding was efficiently inhibited by alpha-bungarotoxin, nicotine, carbachol, and d-tubocurarine. [3H]QNB binding was competitively inhibited by atropine, pirenzepine, and carbachol. Hexamethonium did not affect the binding of either ligand. In competition experiments with [3H]QNB, pirenzepine recognized only one binding site with "low affinity," and carbachol recognized two sites with different affinities. beta-adrenergic receptors were present in a very low amount, whereas alpha-adrenergic and dopaminergic receptors were not detectable. IMR32 cells had an imipramine-sensitive [3H]dopamine uptake, but carbachol, high levels of K+, the calcium ionophore A23187, and alpha-latrotoxin were not able to induce release of [3H]dopamine that had been taken up. The ultrastructural analysis showed that IMR32 cells contained very few dense-core vesicles, suggesting a low storage capacity for neurotransmitter. These cells could be an useful in vitro model for studying neurotransmitter receptors of the human CNS.     

人肺癌细胞系HB-99的建立及其生物学特征

利用一例肺鳞癌手术标本通过原代培养建立了肺癌细胞系命名为HB-99。该细胞系呈单层贴壁生长,从相差显微镜和电镜分析具有细胞的多形性,细胞倍增时间为24小时,克隆形成率40%,染色体改变复杂,众数63-65。将细胞移植到裸鼠体内而生长的肿块具有与原始病人手术标本相似的组织形态。免疫组织化学分析,近100%的细胞表达角蛋白17（CK17）,10%的细胞表达波形蛋白（vimentin）。根据该细胞系的生物学特征提示HB-99是一新建立的肺鳞癌细胞系。 Abstract：We have established a human lung squamous carcinoma cell line ,designed HB-99, by culturing primary tumor sample. The cells of HB-99 derived from resected specimen of a male patient with lung squamous cancer. They grew in monolayers and showed cellular morphology by phase contrast and electronic microscopy. The HB-99 cells had a doubling time of 24 hours and a cloning efficiency of 40%. Chromosomal analysis showed complicated rearrangements with a modal number of 63～65. When hetero-transplanted to nude mice, HB-99 grew to form tumor with the same morphology as the original one from the patient. The results of immunohistochemistry suggested that CK17 expressed in almost all cells while only 5%～10% cells had Vimentin. HB-99 is really a newly established cell line of lung squamous carcinoma.