O-Linked Glycosylation of the Mucin Domain of the Herpes Simplex Virus Type 1-specific Glycoprotein gC-1 Is Temporally Regulated in a Seed-and-spread Manner

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, participating in viral receptor interactions and immunity interference, harbors a mucin-like domain with multiple clustered O-linked glycans. Using HSV-1-infected diploid human fibroblasts, an authentic target for HSV-1 infection, and a protein immunoaffinity procedure, we enriched fully glycosylated gC-1 and a series of its biosynthetic intermediates. This fraction was subjected to trypsin digestion and a LC-MS/MS glycoproteomics approach. In parallel, we characterized the expression patterns of the 20 isoforms of human GalNAc transferases responsible for initiation of O-linked glycosylation. The gC-1 O-glycosylation was regulated in an orderly manner initiated by synchronous addition of one GalNAc unit each to Thr-87 and Thr-91 and one GalNAc unit to either Thr-99 or Thr-101, forming a core glycopeptide for subsequent additions of in all 11 GalNAc residues to selected Ser and Thr residues of the Thr-76–Lys-107 stretch of the mucin domain. The expression patterns of GalNAc transferases in the infected cells suggested that initial additions of GalNAc were carried out by initiating GalNAc transferases, in particular GalNAc-T2, whereas subsequent GalNAc additions were carried out by followup transferases, in particular GalNAc-T10. Essentially all of the susceptible Ser or Thr residues had to acquire their GalNAc units before any elongation to longer O-linked glycans of the gC-1-associated GalNAc units was permitted. Because the GalNAc occupancy pattern is of relevance for receptor binding of gC-1, the data provide a model to delineate biosynthetic steps of O-linked glycosylation of the gC-1 mucin domain in HSV-1-infected target cells.     

Relationship between European eel Anguilla anguilla infection with non‐native parasites and swimming behaviour on encountering accelerating flow

The effect of Anguillicola crassus, Pseudodactylogyrus bini and Pseudodactylogyrus anguillae infection on the behaviour of downstream migrating adult European eels Anguilla anguilla as they encountered accelerating water velocity, common at engineered structures where flow is constricted (e.g. weirs and bypass systems), was evaluated in an experimental flume. The probability of reacting to, and rejecting, the velocity gradient was positively related to A. crassus larval, adult and total abundance. High abundance of Pseudodactylogyrus spp. reduced this effect, but A. crassus was the strongest parasitic factor associated with fish behaviour, and abundance was positively related to delay in downstream passage. Delayed downstream migration at hydraulic gradients associated with riverine anthropogenic structures could result in additional energetic expenditure for migrating A. anguilla already challenged by A. crassus infection.     

构建单纯疱疹病毒基因组Bam HIC片段上有插入突变的重组病毒

单纯疱疹病毒(GSV)是重要的人类DNA病毒。国内外学者正在对其基因结构、功能和基因调控机制进行探索,以及利用其作为病毒载体表达外源基因研制基因工程疫苗等,并且已经取得了一定的成绩。多年来,尽管对HSV-1的分子遗传学做了包括序列分析在内的大量研究,但对HSV基因组许多部位的功能,它们相互之间的关系,特别是某特定部位对活病毒生物特性的作用(病毒生长、繁殖、毒力等),所知很少。为了深入了解HSV-1基因组中Bam H I C片段这一未知部分的功能,我们对它进行了克隆、次级克隆和酶谱分析,并且利用胸腺嘧啶核苷激酶基因—小Mu噬菌体系统(TK-mM)组建了在HSV-1的Eam H I C片段上有TK-mM插入的重组质粒。本文报道利用组建的重组质粒DNA和提纯的TK-HSV-1毒株DNA共同转染细胞,获得了在HSV-1基因组Burn H I C片段上有插入突变的重组病毒。     

Protein arginine methyltransferase 1-directed methylation of Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen

The Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a multifunctional protein with roles in gene regulation and maintenance of viral latency. Post-translational modification of LANA is important for functional diversification. Here, we report that LANA is subject to arginine methylation by protein arginine methyltransferase 1 in vitro and in vivo. The major arginine methylation site in LANA was mapped to arginine 20. This site was mutated to either phenylalanine (bulky hydrophobic, constitutive methylated mimetic) or lysine (positively charged, non-arginine methylatable) residues. The significance of the methylation in LANA function was examined in both the isolated form and in the context of the viral genome through the generation of recombinant KSHV. In addition, authentic LANA binding sites on the KSHV episome in naturally infected cells were identified using a whole genome KSHV tiling array. Although mutation of the methylation site resulted in no significant difference in KSHV LANA subcellular localization, we found that the methylation mimetic mutation resulted in augmented histone binding in vitro and increased LANA occupancy at identified LANA target promoters in vivo. Moreover, a cell line carrying the methylation mimetic mutant KSHV showed reduced viral gene expression relative to controls both in latency and in the course of reactivation. These results suggest that residue 20 is important for modulation of a subset of LANA functions and properties of this residue, including the hydrophobic character induced by arginine methylation, may contribute to the observed effects.     

Effects of Kaposi's sarcoma-associated herpesvirus ORF K1 on differentiating endothelium in murine embryoid bodies and teratomas

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is a prerequisite for Kaposi's sarcoma, a multicentric endothelial tumor of mixed blood vessel-lymphatic phenotype with a marked production of endothelial basement membrane proteins. The viral K1 gene encodes a unique transmembrane protein capable of transforming T lymphocytes experimentally. We studied the effects of K1 regulated by the endothelium-specific tie-1 promoter in the embryonic stem cell (ES)-derived endothelium of subcutaneous murine teratomas and in embryoid body cultures with and without supplemental basic fibroblast growth factor and vascular endothelial growth factor. K1 embryoid bodies showed no difference in endothelial BM protein expression, formation of basal lamina by electron microscopy or fractional area of endothelial growth in comparison with vector controls, but maturation into branching pseudovascular tubes was mildly impaired. Moreover, the marked increase in endothelial fraction induced by both growth factors in the early growth period was absent in K1 clones. Teratoma endothelium, which is partly derived from ES, showed increased proliferation and apoptosis in K1 tumors, coinciding with a possible increased tendency for microhemorrhages. K1 can thus modulate endothelial cell turnover and growth factor response, but does not alter the extracellular matrix of differentiating endothelial cells.     

B virus, Herpesvirus simiae: historical perspective

Between 1932 and 1972, 24 known infections of man by B virus caused 23 cases of encephalitis and 18 fatalities. The virus has been isolated from dermal lesions and neural ganglia from macaque monkeys. Serological evidence of infection is complicated by close antigenic relationships between B virus, Herpesvirus simplex, and SA8. Hyperimmune globulin produced from monkey, horse, and rabbit sera has not proved highly effective. Formalin-inactivated vaccine appears safe and antigenic in man but has not been licensed. Half of all human subjects have neutralizing B virus antibody related to their H. simplex titer. More stable animal populations and the improved use of protective apparel have reduced, but not eliminated, the risk of B virus to man.     

HHV-6体外感染对外周血单个核细胞IL-6、IL-8的诱生NK活性的影响

采用生物活性法和／或酶联免疫吸附法以及乳酸脱氢酶释放法,研究了人类疱疹病毒6型（HHV－6）南京地方株CNS对人外周血单个核细胞（PBMCs）的IL—6、IL－8的诱生和NK活性的影响,并与国外的GS株作比较。结果发现,HHV—6CN8、GS两株病毒感染均可诱导PBMCs产生IL－8,48h达到峰值。两株病毒所诱生的IL－8水平并无显著性差异（P＞0．05）,并可抑制IL－6的产生,但GS株的抑制作用强于CNS株（P＜0．05）。HHV－6体外感染12～24h可以增强NK活性,且CN8株诱导的NK活性高于GS株（P＜0．05）,之后NK活性逐渐减弱。以上结果提示：HHV－6感染可以通过诱生细胞因子和改变NK活性而影响人的免疫功能,而A组的GS株对免疫功能的抑制作用大于B组的CN8株。