Transient Inhibition of Polyoma Virus Synthesis by Sendai Virus (Parainfluenza I) II. Mechanism of the Interference by Inactivated Virus

The mechanism of the transient inhibition of polyoma virus synthesis by betapropiolactone-inactivated Sendai virus was studied. Polyoma virus early functions did not appear to be affected, although deoxyribonucleic acid (DNA) and structural protein synthesis were inhibited 60 and 35% respectively. The inhibition of macromolecular synthesis was not sufficient to account for the 90% inhibition of infectious progeny formation. Encapsidation of polyoma DNA into mature virions appears to be completely inhibited after superinfection by beta-propiolactone-inactivated Sendai virus. Ultraviolet irradiation of live or beta-propiolactone-inactivated Sendai virus preparations abolishes the interfering capacity, indicating that a functional Sendai virus ribonucleic acid molecule is the interfering component.     

Inhibition of the Human Immunodeficiency Virus Type 1 DNA Integration by Modified Oligonucleotides

Oligonucleotide inhibitors of the HIV-1 DNA integration identified to date are reviewed. Two basic strategies of blocking the integration are considered: shielding the integrase-binding sites on the viral DNA by triplex-forming oligonucleotides, and directly inhibiting the enzyme with oligonucleotide agents.     

蛋白质起始的DNA病毒复制机制

DNA聚合酶在DNA合成过程中需要的引物包括RNA引物、DNA自我引物和蛋白质引物3种类型。新DNA链(如冈崎片段)的复制多是在DNA模板上合成一段RNA引物,细小病毒利用其基因组末端的反向末端重复序列(ITRs)自我折叠成DNA引物,而一些DNA、RNA病毒及真菌质粒起始复制反应的引物则是蛋白质。以感染原核生物的噬菌体Phi29和真核DNA病毒腺病毒为例,从复制过程所涉及的蛋白质、对复制原点的识别、复制起始反应、新链的延伸、复制终止过程等方面详细阐述DNA病毒由蛋白质引发的复制机制,并对已商品化的Phi29 DNA聚合酶产品多重置换扩增及单细胞测序等的应用以及基于噬菌体Phi29蛋白质起始的最小复制系统体外扩增异源DNA等最新的应用研究作相关总结介绍。     

Inhibition of Interferons by Ectromelia Virus

Ectromelia virus (EV) is an orthopoxvirus (OPV) that causes mousepox, a severe disease of laboratory mice. Mousepox is a useful model of OPV infection because EV is likely to be a natural mouse pathogen, unlike its close relatives vaccinia virus (VV) and variola virus. Several studies have highlighted the importance of mouse interferons (IFNs) in resistance to and recovery from EV infection, but little is known of the anti-IFN strategies encoded by the virus itself. We have determined that 12 distinct strains and isolates of EV encode soluble, secreted receptors for IFN-gamma (vIFN-gammaR) and IFN-alpha/beta (vIFN-alpha/betaR) that are homologous to those identified in other OPVs. We demonstrate for the first time that the EV vIFN-gammaR has the unique ability to inhibit the biological activity of mouse IFN-gamma. The EV vIFN-alpha/betaR was a potent inhibitor of human and mouse IFN-alpha and human IFN-beta but, surprisingly, was unable to inhibit mouse IFN-beta. The replication of all of the EVs included in our study and of cowpox virus was more resistant than VV to the antiviral effects induced in mouse L-929 cells by IFN-alpha/beta and IFN-gamma. Sequencing studies showed that this EV resistance is likely to be partly mediated by the double-stranded-RNA-binding protein encoded by an intact EV homolog of the VV E3L gene. The absence of a functional K3L gene, which encodes a viral eIF-2alpha homolog, in EV suggests that the virus encodes a novel mechanism to counteract the IFN response. These findings will facilitate future studies of the role of viral anti-IFN strategies in mousepox pathogenesis. Their significance in the light of earlier data on the role of IFNs in mousepox is discussed.     

三链DNA的形成抑制DNA结合蛋白与启动子的结合

电泳迁移分析方法及DNaseⅠ足迹实验表明21nt脱氧寡核苷酸G3TG2T GT2G5TG2TGT(CP1)与乙肝病毒(HBV)核心启动子(Cp)片段之间三链DNA的形成有较高的特异性及稳定性.凝胶滞留实验显示, 在大鼠肝细胞核提取物体外转录系统中, CP1可特异地抑制DNA结合蛋白与Cp片段的结合, 而不能与Cp结合形成三链DNA的脱氧寡核苷酸CP3(TGTG2TG5T2GTG2TG3)对蛋白与Cp的结合并无抑制作用.这些结果表明, 三链DNA的形成有可能抑制HBV DNA的转录.     

Mechanism for Calcium Ion Sensing by the C2A Domain of Synaptotagmin I

The C2A domain is one of two calcium ion (Ca(2+))- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca(2+) sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2A's response to Ca(2+) and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca(2+)- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca(2+)- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I.     

Transient Inhibition of Polyoma Virus Synthesis by Sendai Virus (Parainfluenza I) I. Demonstration and Nature of the Inhibition by Inactivated Virus

Superinfection of polyoma virus-infected mouse embryo cells by beta-propiolactone-inactivated Sendai virus resulted in a 90% inhibition of the synthesis of infectious polyoma progeny. The interference is dependent upon the time of superinfection and the concentration of the inactivated virus. The inhibition of polyoma virus synthesis is transient in nature since normal synthesis of polyoma progeny virus is seen upon prolonged incubation. Interferon does not appear to be implicated in the interference. Various aspects of the biological and synthetic capabilities of beta-propiolactone-inactivated Sendai virus are also described.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.     

Mechanism for Inhibition of Deoxyribonuclease Activity by Antisera

The activity of bovine DNase, but not that of porcine DNase, is inhibited by antisera against bovine DNase, and vice versa. Inhibition of DNase is found in the immunoglobulin G-containing fractions, as shown by ion exchange chromatography. Inactive DNase, carboxymethylated specifically at the active site His¹³⁴, competes with active DNase and reverses the antisera inhibition of DNase, suggesting that the epitode responsible for inhibition does not contain the active site His¹³⁴. Alignment of the sequences of DNase of these two species shows that the greatest variation occurs between residues 153 and 163, within which are three consecutive peptide bonds, Lys-Trp-His-Leu, that are readily cleaved by trypsin, chymotrypsin, or thermolysin. The 8-hr digest of DNase by each of these three proteases has lost the ability to reverse antisera inhibition. The degree of antisera inhibition varies with the metal ion used as the activator for DNase-catalyzed reactions. When Mn²⁺, Co²⁺, or Mg²⁺ plus Ca²⁺ are used as activators, inhibition is approximately 50%. When pBR322 plasmid is used as substrate, gel electrophoresis shows that the DNase-catalyzed DNA hydrolysis produces a significant amount of double-strand cuts with Mn²⁺, Co²⁺, or Mg²⁺ plus Ca²⁺ as activators and antisera inhibit DNase action only on double-strand cuts. With only Mg²⁺ as the activator no double-strand cuts are observed, either in the presence or absence of antisera, and the DNase activity is not significantly inhibited. We conclude that antisera inhibition is due to antibody binding of the DNase polypeptide chain within residues 153 and 163. These residues are not crucial for catalysis, but are required for DNA binding, which results in double-strand cuts.     

致瘤性DNA病毒的整合机制及致瘤效应

恶性肿瘤是一种严重危害人类生命和健康的疾病,而致瘤性DNA病毒是多种恶性肿瘤的主要致病因子.致瘤性DNA病毒的整合可以使宿主细胞正常组织逐步向炎症组织转变,并可导致癌变.病毒整合可引起宿主细胞基因组不稳定和重排,产生新的融合基因,并导致宿主基因表达异常,也是病毒本身得以复制,逃避宿主免疫识别并长期维系自我生存的机制之一.本文综述了目前对致瘤性DNA病毒整合规律以及致瘤性DNA病毒整合致瘤效应的研究和进展,并展望致瘤性DNA病毒整合的研究方向以及在肿瘤发生、发展、诊断和治疗上的应用前景.     

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.     

新型胞质DNA感受通路：cGAS-STING的研究进展

细胞胞质中存在的游离DNA一直被宿主的固有免疫系统当做潜在的危险信号,但免疫系统识别和清除这些危险信号的机制还不明确.近几年有研究发现,DNA感受器(DNA sensor)是宿主感受DNA和免疫防御的桥梁,目前已经有超过10种DNA感受器被发现,而干扰素刺激基因(stimulator of interferon genes,STING,也称为TMEM173、MPYS、MITA和ERIS)作为一种DNA感受通路下游关键的接头分子,在感受胞质DNA和免疫防御方面起着重要的信号传递作用,胞质中的DNA可通过DNA感受器激活STING,再激活Ⅰ型干扰素和其他细胞因子,进而启动机体的免疫反应.近些年,胞质中游离DNA如何激活STING,进而如何在体内启动免疫反应以产生抗病毒或抗菌作用的机制研究取得了显著进展.最近,在哺乳动物细胞中发现了一种新型的核酸转移酶cGAS(cyclic GMP-AMPsynthase),它能识别DNA并能产生一种内源性的环化二核苷酸cGAMP(cyclic GMP-AMP)激活STING.本文将最近关于cGAS的发现、胞质DNA通过cGAS激活STING及STING活化后激活机体免疫反应的机制进行了综述.     

Inhibition in the Joining of DNA Intermediates to Growing Simian Virus 40 Chains

Viral DNA synthesis was inhibited for 1 h by the addition of 5-fluorodeoxyuridine (FUdR) to simian virus 40 (SV40)-infected cultures at 28 to 30 h postinfection. The subsequent addition of (3)H-thymidine to the inhibited cultures reverses the effect of the inhibitor, and during a 1-min labeling period there is rapid synthesis of SV40 DNA. By alkaline sedimentation analysis, it is observed that in FUdR-treated cultures there is synthesis of 4S SV40 DNA intermediates but there is a block in the joining of these intermediates to growing SV40 chain cultures. In addition to 4S fragments that are associated with replicating SV40 molecules, there is accumulation of SV40 DNA in the 6 to 8S region which is observed in neutral sucrose gradients. In an inhibited culture that is pulsed for 1 min with (3)H-thymidine and then chased for 10 min, accumulation of a Component II (Comp. II)-like material is observed. This Comp. II has the same neutral sedimentation characteristics and yields the same R(I) restriction endonuclease product as does authentic Comp. II. However, in alkali it is seen that it is composed of fragmented SV40 DNA. The basis for the failure of 4S fragments to join to growing SV40 chains is discussed. A model in which there is a requirement for two DNA polymerases and a ligase to permit SV40 DNA chain growth is proposed which is consistent with the data presented.     

Preferential Inhibition of Herpes-Group Viruses by Phosphonoacetic Acid: Effect on Virus DNA Synthesis and Virus-Induced DNA Polymerase Activity

In tissue culture phosphonoacetic acid (PAA) specifically inhibited DNA synthesis of human cytomegalovirus (CMV), murine CMV, simian CMV, Epstein-Barr virus, and Herpesvirus saimiri. Fifty to one hundred micrograms per milliliter PAA completely inhibited viral DNA synthesis with no significant damage to host cell DNA synthesis. In vitro DNA polymerization assays showed that 10 μg/ml of PAA specifically inhibited partially purified human CMV-induced DNA polymerase, while little inhibition of host-cell DNA polymerase activity was found. The specific inhibition of herpes-group virus DNA synthesis with little toxicity to host cells suggests that PAA has great potential as an antiherpesvirus therapeutic agent.     

The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition by E4orf4 contributes both to the efficiency of adenovirus replication and to the ability of E4orf4 to kill cancer cells.