人类疱疹病毒7型体外生长特点的研究

研究了人类疱疹病毒7型(HHV-7)南京株YY5在脐血单个核细胞(CBMCs)及SUPT1细胞株上生长特点.观察病毒感染细胞后不同时间病变效应程度,记数死亡细胞百分率,间接免疫荧光染色记数抗原表达阳性细胞数,并用透射电镜观察病毒感染细胞超微结构变化及病毒复制不同时期的特点.结果发现:HHV-7在CBMCs及SUPT1上出现CPE时间迟于HHV-6,且CPE程度也低于HHV-6;HHV-7在SUPT1细胞上尽管有CPE及抗原表达,但很难找到成熟的病毒颗粒;电镜下,病毒主要存在于肿胀的病变细胞内,且病毒感染细胞常出现核染色质聚集,核固缩,及胞浆细胞器空泡化,细胞裂解等特点;成熟的HHV-7颗粒直径约170-190 nm,核衣壳约90-100 nm,核衣壳内致密核心约40 nm,核衣壳与包膜之间是丰富的被膜约30-35 nm,包膜上有刺突.HHV-7常发现无核心的病毒颗粒.     

A Bridge Crosses the Active-Site Canyon of the Epstein-Barr Virus Nuclease with DNase and RNase Activities

Epstein-Barr virus, a double-stranded DNA (dsDNA) virus, is a major human pathogen from the herpesvirus family. The nuclease is one of the lytic cycle proteins required for successful viral replication. In addition to the previously described endonuclease and exonuclease activities on single-stranded DNA and dsDNA substrates, we observed an RNase activity for Epstein-Barr virus nuclease in the presence of Mn²⁺, giving a possible explanation for its role in host mRNA degradation. Its crystal structure shows a catalytic core of the D-(D/E)XK nuclease superfamily closely related to the exonuclease from bacteriophage lambda with a bridge across the active-site canyon. This bridge may reduce endonuclease activity, ensure processivity or play a role in strand separation of dsDNA substrates. As the DNA strand that is subject to cleavage is likely to make a sharp turn in front of the bridge, endonuclease activity on single-stranded DNA stretches appears to be possible, explaining the cleavage of circular substrates.     

Spontaneous malignomas in Tupaia (tree shrew)

Nine spontaneous malignomas of the tree shrew were detected and analysed during an observation period of nine years. The tumours were histopathologically examined and classified. All malignomas developed in imported Tupaia only. From the tumour cells of two different animals new Tupaia herpesviruses were isolated. This is the first. report on spontaneous malignomas of Tupaia in captivity.     

Experimental simian varicella virus infection of St. Kitts vervet monkeys

Experimental simian varicella virus (SVV) infection of St. Kitts vervet monkeys was evaluated as an animal model to investigate human varicella-zoster virus (VZV) infections. During the incubation period, viremia disseminated infectious virus throughout the body via infected peripheral blood lymphocytes (PBLs). A vesicular skin rash in the inguinal area, and on the abdomen, extremities, and face appeared on day 7–10 postinfection. Necrosis and hemorrhage in lung and liver tissues from acutely infected monkeys were evident upon histologic analysis. Recovery from simian varicella was accompanied by a rise in the serum neutralizing antibody response to the virus. SVV latency was established in trigeminal ganglia of monkeys which resolved the acute infection. This study indicates that experimental SVV infection of St. Kitts vervets is a useful animal model to investigate SVV and VZV pathogenesis and to evaluate potential antiviral agents and vaccines.     

KSHV‐TK is a tyrosine kinase that disrupts focal adhesions and induces Rho‐mediated cell contraction

Paradoxically, the thymidine kinase (TK) encoded by Kaposi sarcoma‐associated herpesvirus (KSHV) is an extremely inefficient nucleoside kinase, when compared to TKs from related herpesviruses. We now show that KSHV‐TK, in contrast to HSV1‐TK, associates with the actin cytoskeleton and induces extensive cell contraction followed by membrane blebbing. These dramatic changes in cell morphology depend on the auto‐phosphorylation of tyrosines 65, 85 and 120 in the N‐terminus of KSHV‐TK. Phosphorylation of tyrosines 65/85 and 120 results in an interaction with Crk family proteins and the p85 regulatory subunit of PI3‐Kinase, respectively. The interaction of Crk with KSHV‐TK leads to tyrosine phoshorylation of this cellular adaptor. Auto‐phosphorylation of KSHV‐TK also induces a loss of FAK and paxillin from focal adhesions, resulting in activation of RhoA‐ROCK signalling to myosin II and cell contraction. In the absence of FAK or paxillin, KSHV‐TK has no effect on focal adhesion integrity or cell morphology. Our observations demonstrate that by acting as a tyrosine kinase, KSHV‐TK modulates signalling and cell morphology.     

The human herpesvirus 6 U21–U24 gene cluster is dispensable for virus growth

Human herpesvirus 6 (HHV‐6) is a T‐lymphotrophic virus belongs to the genus Roseolovirus within the beta herpesvirus subfamily. The U20–U24 gene cluster is unique to Roseoloviruses; however, both their function and whether they are essential for virus growth is unknown. Recently, bacterial artificial chromosome (BAC) techniques have been used to investigate HHV‐6A. This study describes generation of a virus genome lacking U21–U24 (HHV‐6ABACΔU21–24) and shows that infectious virus particles can be reconstituted from this BAC DNA. Our data indicate that the HHV‐6 U21–U24 gene cluster is dispensable for virus propagation.     

Cytokines, macrophage lipid metabolism and foam cells: implications for cardiovascular disease therapy

Cardiovascular disease is the biggest killer globally and the principal contributing factor to the pathology is atherosclerosis; a chronic, inflammatory disorder characterized by lipid and cholesterol accumulation and the development of fibrotic plaques within the walls of large and medium arteries. Macrophages are fundamental to the immune response directed to the site of inflammation and their normal, protective function is harnessed, detrimentally, in atherosclerosis. Macrophages contribute to plaque development by internalizing native and modified lipoproteins to convert them into cholesterol-rich foam cells. Foam cells not only help to bridge the innate and adaptive immune response to atherosclerosis but also accumulate to create fatty streaks, which help shape the architecture of advanced plaques. Foam cell formation involves the disruption of normal macrophage cholesterol metabolism, which is governed by a homeostatic mechanism that controls the uptake, intracellular metabolism, and efflux of cholesterol. It has emerged over the last 20 years that an array of cytokines, including interferon-γ, transforming growth factor-β1, interleukin-1β, and interleukin-10, are able to manipulate these processes. Foam cell targeting, anti-inflammatory therapies, such as agonists of nuclear receptors and statins, are known to regulate the actions of pro- and anti-atherogenic cytokines indirectly of their primary pharmacological function. A clear understanding of macrophage foam cell biology will hopefully enable novel foam cell targeting therapies to be developed for use in the clinical intervention of atherosclerosis.     

Molecular basis for herpesvirus entry mediator recognition by the human immune inhibitory receptor CD160 and its relationship to the cosignaling molecules BTLA and LIGHT

CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4⁺ T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function.     

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (λ) scaffolding protein gpNu3. The purified protein possesses significant α-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition (T_m = 40.6 ± 0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer-dimer equilibrium (K_d,app ~ 50 μM) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the λ major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.