Characterization of Influenza Virus Neuraminidases: Peptide Changes Associated with Antigenic Divergence Between Early and Late N2 Neuraminidases

Neuraminidases (EC 3.2.1.18) of 1957, 1960, and 1969 influenza virus strains were isolated after proteolytic digestion of viral hemagglutinin. Each neuraminidase was recovered with a final yield of about 15% and had similar specific activities. Immunization of rabbits with the neuraminidases elicited monospecific neuraminidase antibodies, with no antibodies to viral hemagglutinin. Further evidence of purity was the existence of only a single component, about 50,000 daltons in size, when reduced neuraminidase preparations were examined by sodium dodecyl sulfate acrylamide gel electrophoresis. However, storage of neuraminidase in solution resulted in the appearance of slightly smaller degradation products. Preparations of each neuraminidase were denatured under reducing conditions, and exposed sulfhydryl residues were blocked by reaction with (14)C-iodoacetamide. After tryptic digestion, peptide maps were prepared for the neuraminidases, and the (14)C-labeled cysteinyl peptides were then identified by autoradiography. About 20 peptides were present, in agreement with the number predicted from amino acid analysis for neuraminidase subunits of only one type. The 1957 and 1960 neuraminidases exhibited a small antigenic divergence from each other, and maps of their cysteinyl peptides appeared to be identical. The 1969 neuraminidase exhibited considerable antigenic divergence from the other two neuraminidases, and maps of 1969 neuraminidase peptides revealed two major and several minor differences from the other maps. Thus, antigenic divergence between the neuraminidases of Asian and Hong Kong influenza viruses is associated with a small number of changes in the primary structure of the neuraminidase subunit.     

Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity in Cells Infected with Influenza Virus

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase activity was assayed on nuclear preparations of chick embryo fibroblast cells at various times after infection with an influenza A virus (fowl plague virus) and was compared with the activity of uninfected cells. Polymerase activity was increased by about 60% by 2 hr after infection, and this increase coincided with an increase in RNA synthesis in infected cells, as determined by pulse-labeling with uridine. No difference could be detected between the polymerases of infected and uninfected cells as to their requirements for DNA primer, divalent cations, and nucleoside triphosphates, and they were equally sensitive to addition of actinomycin D to the reaction mixture. It is possible that host cell DNA-dependent RNA polymerase is involved in the replication of influenza virus RNA.     

唾液酸受体并非流感病毒各亚型在雪貂组织中播散分布的决定因子

目的探讨流感病毒在雪貂组织中的分布与唾液酸受体的关系。方法用病毒分离的方法分析流感病毒H5N1（SZ406H,A/VN/1203/04）,SH1N1,H3N2（Brisbane/09,HK/09）在雪貂各组织中分布,用直接免疫荧光法分析雪貂各组织的唾液酸受体的分布,并通过体外实验证实活病毒与组织上受体的结合。结果 H5N1（SZ）和H5N1（A/VN/1203/04）在雪貂的肝、脾、肺、肠中有分布,H5N1（A/VN/1203/04）在脑组织中也有分布,而SH1N1、H3N2（Brisbane/09,HK/09）只分布于肠组织。而唾液酸受体SAα2,6Gal和SAα2,3Gal的I型受体分布于脾、心、肺、肠、脑组织中,和SAα2,3Gal II型受体分布于肝、脾、心、肺、肠、脑组织。SH1N1病毒与SAα2,6Gal能结合,而H5N1与SAα2,3Gal结合。结论 H5N1能在雪貂的多器官组织组织中分布和繁殖,而H3N2和SH1N1仅能在肠组织中分布繁殖。SAα2,6Gal和SAα2,3Gal受体在雪貂多器官组织中均有表达,说明唾液酸受体是病毒进入的门户,但不是病毒分布的决定因子。     

流感病毒识别糖链受体分子机制的研究进展

近年来,由于流感病毒(influenza virus)不可预测的局部流行和有可能引发全球大流行,其一直是研究的热点课题之一．流感病毒表面糖蛋白血凝素(hemagglutinin,HA)特异识别宿主细胞表面的糖链受体是流感病毒感染宿主、进而复制并继续传播的生物学基础．影响流感病毒宿主特异性的两个主要因素是HA自身的变化(包括基因突变、重组、糖基化位点数量和糖基化位置的变化)和宿主细胞表面糖链受体的变化(包括糖链受体的类型、分布和分子构象的改变)等．因此准确掌握这些信息有助于人们进一步加强对流感病毒的防控．本文主要从糖组学角度概述了流感病毒识别糖链受体的分子机制,重点介绍流感病毒宿主细胞表面糖链受体的研究进展．     

Ribonucleic Acid Synthesis in Cells Infected with Influenza Virus

Virus-specific ribonucleic acid (RNA), synthesized in influenza virus-infected cells from 3.5 to 7.5 hr after infection, was studied. After velocity centrifugation in sucrose, three peaks of virus-specific RNA could be identified: 34S, 18S, and 11S. These RNA species are predominantly single-stranded and consist of 90% viral (plus) and 10% complementary (minus) RNA strands. Most (75%) of the complementary RNA is single-stranded, i.e., not part of RNA duplexes or replicative intermediates. The 34S RNA species is an aggregate of 18S and 14S RNA species. Both 18S and 11S RNA species are relatively heterogenous compared to 18S ribosomal RNA, and these species probably contain different RNA molecules having closely related sedimentation coefficients.     

Acid Sensitivity of the Influenza Virus Hemagglutinin

Influenza virus hemagglutinin was shown to be acid resistant if precipitates which form during acidification are first removed. Adsorption of virus to precipitates formed during acidification may cause a virus to be described incorrectly as acid sensitive.     

Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.     

Force Spectroscopy Shows Dynamic Binding of Influenza Hemagglutinin and Neuraminidase to Sialic Acid

The influenza A virus infects target cells through multivalent interactions of its major spike proteins, hemagglutinin (HA) and neuraminidase (NA), with the cellular receptor sialic acid (SA). HA is known to mediate the attachment of the virion to the cell, whereas NA enables the release of newly formed virions by cleaving SA from the cell. Because both proteins target the same receptor but have antagonistic functions, virus infection depends on a properly tuned balance of the kinetics of HA and NA activities for viral entry to and release from the host cell. Here, dynamic single-molecule force spectroscopy, based on scanning force microscopy, was employed to determine these bond-specific kinetics, characterized by the off rate k_off, rupture length x_β and on rate k_on, as well as the related free-energy barrier ΔG and the dissociation constant K_D. Measurements were conducted using surface-immobilized HA and NA of the influenza A virus strain A/California/04/2009 and a novel, to our knowledge, synthetic SA-displaying receptor for functionalization of the force probe. Single-molecule force spectroscopy at force loading rates between 100 and 50,000 pN/s revealed most probable rupture forces of the protein-SA bond in the range of 10–100 pN. Using an extension of the widely applied Bell-Evans formalism by Friddle, De Yoreo, and co-workers, it is shown that HA features a smaller x_β, a larger k_off and a smaller ΔG than NA. Measurements of the binding probability at increasing contact time between the scanning force microscopy force probe and the surface allow an estimation of K_D, which is found to be three times as large for HA than for NA. This suggests a stronger interaction for NA-SA than for HA-SA. The biological implications in regard to virus binding to the host cell and the release of new virions from the host cell are discussed.     

Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity

We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity.     

Influenza Nucleoprotein Delivered with Aluminium Salts Protects Mice from an Influenza A Virus That Expresses an Altered Nucleoprotein Sequence

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes.     

Mapping of a Region of the PA-X Protein of Influenza A Virus That Is Important for Its Shutoff Activity

Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.