Effect of Envelope Proteins on the Mechanical Properties of Influenza Virus

The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm⁻¹) than a viral protein-capsid coat and sustains deformations of one-third of the virion''s diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host.     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用.结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用.体内实验中0.1 g/kg,0.5g/kg,1.2g/kg 3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间;降低肺炎小鼠的肺指数和血凝滴度(P<0.01).其作用与病毒唑相当.结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂.     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用。结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用。体内实验中0.1g/kg,0.5g/kg,1.2g/kg3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间:降低肺炎小鼠的肺指数和血凝滴度(P<0.01)。其作用与病毒唑相当。结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂。     

Electrophoretic Distribution of the Proteins and Glycoproteins of Influenza Virus and Sendai Virus

The proteins of influenza (WSN) and Sendai virus have been separated by polyacrylamide gel electrophoresis into five components. In both cases, three of these components were shown to be glycoproteins containing fucose, galactose, and glucosamine. Two protein components of each virus were probably free from these sugar residues, including the structural unit of the viral ribonucleoprotein (molecular weight of about 60,000 daltons).     

Time Course of Synthesis and Assembly of Influenza Virus Proteins

The synthesis of viral polypeptides was analyzed in BHK-21-F cells infected with the WSN strain of influenza virus at various times in the growth cycle. The relative amounts of polypeptides P, HA, NP, and NS did not change markedly between early and late times in the growth cycle; however, there was a progressive increase in the relative amount of the M polypeptide at later time points. In cell fractionation experiments, the patterns of newly synthesized polypeptides associated with various cytoplasmic fractions remained similar throughout the growth cycle except for an increase in polypeptide M in all fractions late in the growth cycle. The HA polypeptide was chased out of cytoplasmic membranes completely 6 h after synthesis, whereas the M polypeptide was not chased effectively from such membranes. Marked differences were found in the incorporation into mature virions of polypeptides synthesized at different times in the growth cycle. Polypeptides P and NP synthesized at early times were incorporated preferentially, whereas M was synthesized and incorporated predominantly late in the growth cycle. The fact that the rates of incorporation of polypeptides into virions differed significantly from their rates of synthesis indicates that different polypeptides were assembled into virions by distinct pathways.     

Differences in Dispersion of Influenza Virus Lipids and Proteins during Fusion

Digitally enhanced low-light-level fluorescence video microscopy and immunochemical staining were used to examine influenza virus envelope lipid and protein redistribution during pH-induced fusion. Video microscopy was performed using viruses labeled with either the lipid analogue octadecylrhodamine B (R18) or fluorescein isothiocyanate (FITC) covalently linked to envelope proteins. Viruses were bound to human red blood cells, and the pattern and intensity of fluorescence were monitored for 30 min while cell-virus complexes were perfused with pH 7.4 or 4.8 media at temperatures either above or below 20°C. R18 showed complete redistribution and dequenching by 30 min at all incubation temperatures, confirming reports that viral fusion occurs at subphysiological temperatures. FITC-labeled protein showed spatial redistribution at 28°C but no change at low temperature. Electron microscopy observations of immunochemical staining of viral proteins confirmed both that protein redistribution at 37°C was slower than R18 and the failure of movement within 30 min at 16°C. Video microscopy monitoring of RNA staining by acridine orange of virus-cell complexes showed redistribution to the RBCs at all temperatures but only after low pH-induced fusion. The results are consistent with differential dispersion of viral components into the RBC and the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation.     

Extracellular Matrix Proteins in Hemostasis and Thrombosis

The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell–matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets.The balance between hemostasis and thrombosis relies on a finely tuned adhesive response of blood platelets. Inadequate adhesion leads to bleeding, whereas excessive or inappropriate adhesion leads to thrombosis. Resting platelets are nonadhesive anuclear discs and do not interact with the vessel wall, but they have a plethora of receptors that sense activating signals (agonists) of various sorts. The activating signals include soluble factors such as thrombin, adenosine diphosphate (ADP), and epinephrine, all of which act on G-protein-coupled receptors (GPCRs) on the platelets. In addition, certain receptors for extracellular matrix (ECM) proteins (e.g., GPIb, GPVI, and some integrins) can also act as activating receptors. These diverse receptors trigger intracellular signaling pathways that activate (1) actin assembly leading to cell shape change and extension of filopodia; (2) exocytosis of secretory granules that release additional platelet agonists as well as adhesive ECM proteins; and (3) activation of additional cell-surface receptors such as the major platelet-specific integrin, αIIbβ3, that contribute further to the adhesion and aggregation of activated platelets. Thus, the interactions of platelet-ECM adhesion receptors with ECM proteins from the vessel wall, from the plasma, and from the platelets themselves, are central to both the initial adhesion and the subsequent activation and aggregation of platelets (Varga-Szabo et al. 2008). These adhesive interactions, together with coagulation (to which platelets also contribute), generate the fibrin clot, essentially a facultative ECM that forms the initial occlusion of the damaged vessel but also serves as a subsequent ECM substrate for wound healing. In this article, we will review what is known about the roles of ECM proteins and their receptors in platelet adhesion and aggregation, summarize the roles of the clot and provisional ECM in subsequent wound healing, point out various unanswered questions, and discuss briefly the contributions of the relevant cell–ECM interactions to disease and the potential for therapeutic interventions.     

Swine Interferon-Inducible Transmembrane Proteins Potently Inhibit Influenza A Virus Replication

Human interferon-inducible transmembrane proteins (IFITMs) were identified as restriction factors of influenza A virus (IAV). Given the important role of pigs in the zoonotic cycle of IAV, we cloned swine IFITMs (swIFITMs) and found two IFITM1-like proteins, one homologue of IFITM2, and a homologue of IFITM3. We show that swIFITM2 and swIFITM3 localize to endosomes and display potent antiviral activities. Knockdown of swIFITMs strongly reduced virus inhibition by interferon, establishing the swIFITMs as potent restriction factors in porcine cells.     

Characterization of CELO Virus Proteins That Modulate the pRb/E2F Pathway

The avian adenovirus CELO can, like the human adenoviruses, transform several mammalian cell types, yet it lacks sequence homology with the transforming, early regions of human adenoviruses. In an attempt to identify how CELO virus activates the E2F-dependent gene expression important for S phase in the host cell, we have identified two CELO virus open reading frames that cooperate in activating an E2F-inducible reporter system. The encoded proteins, GAM-1 and Orf22, were both found to interact with the retinoblastoma protein (pRb), with Orf22 binding to the pocket domain of pRb, similar to other DNA tumor virus proteins, and GAM-1 interacting with pRb regions outside the pocket domain. The motif in Orf22 responsible for the pRb interaction is essential for Orf22-mediated E2F activation, yet it is remarkably unlike the E1A LxCxD and may represent a novel form of pRb-binding peptide.     

乙型肝炎病毒蛋白和绿色荧光蛋白的共表达研究

目的：构建增强型绿色荧光蛋白（EGFP）标记的乙型肝炎病毒（HBV）真核表达载体,并研究其在真核细胞和小鼠体内的共表达。方法：以质粒pBR322-HBVadr2．0和pCX-EGFP为基础,构建含有双拷贝HBV全基因组DNA和EGFP基因的真核表达载体pCX-EGFP-HBVadr2．0,分别转染真核细胞和小鼠肝组织,建立体外、体内表达系统,研究GFP和HBV基因的表达。结果：构建了真核表达载体pCX-EGFP-HBVadr2．0,EGFP和HBV病毒蛋白在体内和体外均可表达。结论：构建的pCX-EGFP-HBVadr2．0真核表达载体可以GFP作为HBV存在与否的报告基因,提高了培育检测转基因小鼠的效率,为转基因小鼠的制备及后续研究奠定了基础。     

Antigenic Characterization of Recombinant Hemagglutinin Proteins Derived from Different Avian Influenza Virus Subtypes

Since the advent of highly pathogenic variants of avian influenza virus (HPAIV), the main focus of avian influenza research has been the characterization and detection of HPAIV hemagglutinin (HA) from H5 and H7 subtypes. However, due to the high mutation and reassortation rate of influenza viruses, in theory any influenza strain may acquire increased pathogenicity irrespective of its subtype. A comprehensive antigenic characterization of influenza viruses encompassing all 16 HA and 9 neuraminidase subtypes will provide information useful for the design of differential diagnostic tools, and possibly, vaccines. We have expressed recombinant HA proteins from 3 different influenza virus HA subtypes in the baculovirus system. These proteins were used to generate polyclonal rabbit antisera, which were subsequently employed in epitope scanning analysis using peptide libraries spanning the entire HA. Here, we report the identification and characterization of linear, HA subtype-specific as well as inter subtype-conserved epitopes along the HA proteins. Selected subtype-specific epitopes were shown to be suitable for the differentiation of anti-HA antibodies in an ELISA.     

用表面等离子体共振技术筛选与禽流感病毒基质蛋白M1相互作用的蛋白

目的：筛选与禽流感病毒基质蛋白M1相互作用的蛋白。方法：表达禽流感病毒基质蛋白M1,经Ni^2＋柱亲和层析纯化,用表面等离子体共振（SPR）技术捕获BHK-21细胞裂解液中与M1相互作用的细胞蛋白,并进行质谱分析。结果：获得了纯度在85%以上的基质蛋白M1,并利用此蛋白捕获到宿主细胞肌球蛋白重链6。结论：肌球蛋白重链6与禽流感病毒基质蛋白M1可能存在体外相互作用。     

Previously Unrecognized Amino Acid Substitutions in the Hemagglutinin and Fusion Proteins of Measles Virus Modulate Cell-Cell Fusion,Hemadsorption, Virus Growth,and Penetration Rate

Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.Measles virus (MV), which belongs to the genus Morbillivirus in the family Paramyxoviridae, is an enveloped virus with a nonsegmented negative-strand RNA genome. The MV genome encodes six structural proteins: the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large (L) proteins. The P gene also encodes two other accessory proteins, the C and V proteins. The C protein is translated from an alternative translational initiation site leading a different reading frame, and the V protein is synthesized from an edited mRNA. MV has two envelope glycoproteins, the F and H proteins. The former is responsible for envelope fusion, and the latter is responsible for receptor binding (12).Wild-type MV strains isolated in B95a cells and laboratory-adapted MV strains have distinct phenotypes (18). Wild-type MV strains can grow in B95a cells but not in Vero cells, while laboratory-adapted MV strains can grow in both B95a and Vero cells. Wild-type MV strains do not cause hemadsorption (HAd) in African green monkey red blood cells (AGM-RBC), while most of laboratory-adapted MV strains cause HAd. Importantly, wild-type MV strains are pathogenic and induce clinical signs that resemble human measles in experimentally infected monkeys while laboratory-adapted MV strains do not.One approach to identify amino acid substitutions responsible for these phenotypic differences is the comparison of a wild-type MV strain with a standard laboratory-adapted MV strain such as the Edmonston strain. With regard to the H protein, amino acid substitutions important for HAd activity and cell-cell fusion in tissue culture cells were identified by expressing the H proteins in mammalian cells (15, 21). Recently, Tahara et al. revealed that the M, H, and L proteins are responsible for efficient growth in Vero cells by constructing a series of recombinant viruses in which part of the genome of the wild-type MV was replaced with the corresponding sequences of the Edmonston strain (45, 46, 47).Another approach is the comparison of wild-type MV strains with their Vero cell-adapted MV strains. It was reported that Vero cell-adapted MV strains could be obtained by successive blind passages of wild-type MV strains in Vero cells (18, 24, 30, 43). Interestingly, in vivo and in vitro phenotypes of Vero cell-adapted MV strains were similar to those of laboratory-adapted standard MV strains (18, 19, 24, 30, 43). Comparison of the complete nucleotide sequences of the genomes of wild-type MV strains with those of Vero cell-adapted wild-type MV strains revealed amino acid substitutions in the P, C, V, M, H, and L proteins (27, 42, 48, 53).At present, these phenotypic differences are explained mainly by the receptor usage of MV. Wild-type MV strains can use signaling lymphocyte activation molecule (SLAM; also called CD150) but not CD46 as a cellular receptor, whereas laboratory-adapted MV strains can use both SLAM and CD46 as cellular receptors (7, 10, 16, 29, 56, 60).However, receptor usage per se cannot explain all of the phenotypic differences (20, 25, 48, 53). For example, recombinant Edmonston strains expressing wild-type H proteins can grow in Vero cells to some extent (17, 54). Several reports suggested the presence of the third MV receptor on Vero cells (14, 44, 54, 60). Other reports indicated the contribution of the M protein on cell-cell fusion and growth of MV in Vero cells (4, 27, 47). Recently, the unidentified epithelial cell receptor for MV was predicted in primary culture of human cells (1, 55) and several epithelial cell lines (23, 51). However, the identity of the third receptor on Vero cells and the unidentified epithelial cell receptor is not clear yet. Thus, the mechanism of Vero cell adaptation of wild-type MV is not completely understood.In order to understand the molecular mechanism of these phenotypic changes of wild-type MV strains during adaptation in Vero cells, we determined the complete nucleotide sequences of the genomes of the wild-type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strains (43) and examined the effect of individual amino acid substitutions using a mammalian cell expression system and reverse genetics. We show here that previously unrecognized new amino acid substitutions in the H and F proteins are important for MV adaptation and HAd activity.     

IFN和PDS合剂体内抗流感病毒的作用

观察了IFN与PDS合剂的体内抗病毒效果。首先排除IFN和PDS对动物的毒性作用 ,再以不同剂量的单剂和合剂给小鼠用药。用流感强毒株对小鼠进行攻击 ,同时以利巴韦林和正常小鼠作为对照。通过对动物死亡率、肺部病变的病理组织学检查结果进行比较 ,合剂组效果明显好于单剂组 ,且有剂量效应关系。上述结果表明 ,IFN和PDS合剂可明显减轻流感病毒感染过程中的肺组织病变 ,可用于流行性感冒的治疗。     

Influenza B and C Virus NEP (NS2) Proteins Possess Nuclear Export Activities

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.