黄热病毒的基因组及其蛋白研究进展

黄热病毒(Yellowfever virus,YFV)是属于黄病毒科(Flavivirdae)黄病毒属(Flavivirus)的典型代表,为RNA病毒其不仅是第一个被发现的导致人类疾病的"滤过性颗粒",也是第一个被证实通过蚊蜱传播的病毒。黄热病是一种区域性疾病,在南美     

Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus

The in vivo mechanism for virulence attenuation of laboratory-derived variants of two flaviviruses in the Japanese encephalitis virus (JEV) serocomplex is described. Host cell adaptation of JEV and Murray Valley encephalitis virus (MVE) by serial passage in adenocarcinoma cells selected for variants characterized by (i) a small plaque phenotype, (ii) increased affinity to heparin-Sepharose, (iii) enhanced susceptibility to inhibition of infectivity by heparin, and (iv) loss of neuroinvasiveness in a mouse model for flaviviral encephalitis. We previously suggested that virulence attenuation of the host cell-adapted variants of MVE is a consequence of their increased dependence on cell surface glycosaminoglycans (GAGs) for attachment and entry (E. Lee and M. Lobigs, J. Virol. 74:8867-8875, 2000). In support of this proposition, we find that GAG-binding variants of JEV and MVE were rapidly removed from the bloodstream and failed to spread from extraneural sites of replication into the brain. Thus, the enhanced affinity of the attenuated variants for GAGs ubiquitously present on cells and extracellular matrices most likely prevented viremia of sufficient magnitude and/or duration required for virus entry into the brain parenchyma. This mechanism may also account, in part, for the attenuation of the JEV SA14-14-2 vaccine, given the sensitivity of the virus to heparin inhibition. A pronounced loss of the capacity of the GAG-binding variants to produce disease was also noted in mice defective in the alpha/beta interferon response, a mouse strain shown here to be highly susceptible to infection with JEV serocomplex flaviviruses. Despite the close genetic relatedness of JEV and MVE, the variants selected for the two viruses were altered at different residues in the envelope (E) protein, viz., Glu(306) and Asp(390) for JEV and MVE, respectively. In both cases the substitutions gave the protein an increased net positive charge. The close spatial proximity of amino acids 306 and 390 in the predicted E protein structure strongly suggests that the two residues define a receptor-binding domain involved in virus attachment to sulfated proteoglycans.     

Production of pseudoinfectious yellow fever virus with a two-component genome

Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.     

发热伴血小板减少综合征布尼亚病毒结构和非结构蛋白表达研究

发热伴血小板减少综合征布尼亚病毒(SFTSV)是我国2010年新发现的新布尼亚病毒,可导致人类严重发热伴血小板减少综合征。SFTS新布尼亚病毒全基因组已解析,但病毒分子生物学结构蛋白特征及功能尚需更多研究。本文通过蔗糖密度梯度离心确定发热伴血小板减少综合征布尼亚病毒(HB29株)病毒颗粒的沉降密度及超离纯化条件,得出该病毒颗粒在蔗糖中的沉降密度为1.135g/mL。利用PCR方法扩增SFTSV病毒株HB29株病毒RNA聚合酶(RdRp)、糖蛋白前体蛋白(M)、包膜糖蛋白(Gn)、包膜糖蛋白(Gc)、核蛋白(NP)及非结构蛋白(NSs)的编码区基因片段,分别克隆入真核表达载体pcDNA5/FRT或VR1012,在293T细胞上获得上述基因表达。通过SDS-PAGE分析纯化病毒颗粒和重组蛋白,并通过免疫印迹(Western blotting)和间接免疫荧光(IFA)确定蛋白活性和分子量。本研究结果将有利于对新布尼亚病毒分子生物学特征的认识,为后期研究提供基础。     

Role of antibodies and host cells in plaque enhancement of Murray Valley encephalitis virus.

Chicken antisera to Murray Valley encephalitis (MVE) virus, when incubated with virus and assayed for plaques on chicken embryo (CE) monolayers, neutralized MVE virus at high concentrations of antibody, but caused increases in plaque counts at low concentrations of antibody. Plaque enhancement did not occur when the same virus-antibody mixtures were assayed on a continuous line of rhesus monkey kidney cells (LLC-MK2), nor when the anti-MVE antibody was of mammalian origin and the assay system was CE monolayers. Correspondingly, chicken anti-MVE did not enhance the plaque formation of MVE virus in a stable line of mouse macrophages, P-388D1, whereas rabbit and mouse anti-MVE did enhance plaque formation. This enhancing activity was associated with noncytophilic immunoglobulin G (IgG). The Fc terminus of the IgG molecule was required, as no plaque enhancement occurred with chicken anti-MVE Fab. These data indicate that there is a requirement for taxonomic complementarity between Fc termini and Fc receptors in the above systems. CE cell monolayers were found to contain approximately 2% of Fc receptor-bearing cells among the fibroblast-like cells. Fc receptor-bearing cells in CE monolayers were isolated and found to be of the mononuclear phagocytic lineage. These mononuclear phagocytes, which originate in lymphoid tissues and blood associated with CE tissue fragments, are integrated into primary CE monolayers and form infectious centers in the presence of virus and low dilutions of antibody.     

Structure of the Murray Valley encephalitis virus RNA helicase at 1.9 Angstrom resolution

Murray Valley encephalitis virus (MVEV), a mosquito-borne flavivirus endemic to Australia, is closely related to Japanese encephalitis virus and West Nile virus. Nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains, whose activity is central to flavivirus replication and is therefore a possible target for anti-flaviviral compounds. Cloning, purification, and crystal structure determination to 1.9 Angstrom resolution of the NS3 helicase of MVEV and characterization of its enzymatic activity is reported. Comparison with the structures of helicases from related viruses supports a possible mechanism of ATP hydrolysis-driven strand separation.     

Murray Valley encephalitis virus infection in mosquitoes and domestic fowls in Queensland, 1974.

Field studies during an epidemic of Murray Valley encephalitis (MVE) led to the isolation of MVE virus from a pool of mosquitoes (Culex annulirostris) and a sentinel chicken from Charleville, south-west Queensland. A high proportion of domestic fowls at Charleville had antibody to MVE virus at the beginning of February 1974, in advance of the first case recognized in Queensland and allowing early warning from health authorities. A survey of antibody in domestic fowls in mid-1974 suggested widespread activity of MVE virus in western and east-central Queensland. Virus isolation and serological studies showed activity in south-west Queensland of three other viruses known to infect man, Ross River, Sindbis and Kunjin viruses.     

Serological evidence of inter-epidemic infection of feral pigs in New South Wales with Murray Valley encephalitis virus.

The sera of 617 feral pigs, collected from three widely separated areas of northern and central New South Wales, were examined for antibody to Murray Valley encephalitis (MVE) virus and to Ross River virus. Haemagglutination-inhibition (HI) antibody was detected to MVE in 58% of sera and to Ross River virus in 15% of sera. Neutralization tests suggested that the MVE HI antibody resulted from infection with MVE virus in the summers of 1971-1972 and 1972-1973 when the virus was not known to be active in New South Wales. These same tests suggested that more than one flavivirus infected the feral pigs in the summer of 1973-1974 and that Kunjin virus was active in the summer of 1975-1976.     

The nucleotide sequence of cDNA coding for the structural proteins of foot-and-mouth disease virus

The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.     

The complete nucleotide sequence of the maize chlorotic mottle virus genome.

The complete nucleotide sequence of the maize chlorotic mottle virus (MCMV) genome has been determined to be 4437 nucleotides. The viral genome has four long open reading frames (ORFs) which could encode polypeptides of 31.6, 50, 8.9 and 25.1 kd. If the termination codons, for the polypeptides encoded by the 50 and 8.9 kd ORFs are suppressed, readthrough products of 111 and 32.7 kd result. The 31.6 and 50 kd ORFs overlap for nearly the entire length of the 31.6 kd ORF. Striking amino acid homology has been observed between two potential polypeptides encoded by MCMV and polypeptides encoded by carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 25.1 kd ORF most likely encodes the capsid protein. The similar genome organization and amino acid sequence homology of MCMV with CarMV and TCV suggest an evolutionary relationship with these members of the carmovirus group.     

Creation of a recombinant Rift Valley fever virus with a two-segmented genome

Rift Valley fever virus (RVFV; family Bunyaviridae) is a clinically important, mosquito-borne pathogen of both livestock and humans, which is found mainly in sub-Saharan Africa and the Arabian Peninsula. RVFV has a trisegmented single-stranded RNA (ssRNA) genome. The L and M segments are negative sense and encode the L protein (viral polymerase) on the L segment and the virion glycoproteins Gn and Gc as well as two other proteins, NSm and 78K, on the M segment. The S segment uses an ambisense coding strategy to express the nucleocapsid protein, N, and the nonstructural protein, NSs. Both the NSs and NSm proteins are dispensable for virus growth in tissue culture. Using reverse genetics, we generated a recombinant virus, designated r2segMP12, containing a two-segmented genome in which the NSs coding sequence was replaced with that for the Gn and Gc precursor. Thus, r2segMP12 lacks an M segment, and although it was attenuated in comparison to the three-segmented parental virus in both mammalian and insect cell cultures, it was genetically stable over multiple passages. We further show that the virus can stably maintain an M-like RNA segment encoding the enhanced green fluorescent protein gene. The implications of these findings for RVFV genome packaging and the potential to develop multivalent live-attenuated vaccines are discussed.     

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.