Cassetti M C, Merchlinsky M, Wolffe E J, Weisberg A S, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Virol. 1998 Jul;72(7):5769-80. doi: 10.1128/JVI.72.7.5769-5780.1998.
The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphate-binding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-beta-D-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early- and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging.
痘苗病毒A32开放阅读框预计编码一种具有三磷酸核苷结合基序且质量为34 kDa的蛋白质。为了研究该蛋白质的作用，我们构建了一个突变体，其中原始的A32基因被一个可诱导的拷贝所取代。重组病毒vA32i具有条件致死表型：感染性病毒的形成依赖于异丙基-β-D-硫代半乳糖苷（IPTG）。在非允许条件下，该突变体合成早期和晚期病毒蛋白以及被加工成单位长度基因组的病毒DNA。在没有IPTG的情况下对感染细胞进行电子显微镜观察，发现有外观正常的月牙形和未成熟病毒颗粒，但很少有含类核的颗粒。没有具有明确核心结构的砖形成熟颗粒，而是有许多电子致密的球形颗粒。其中一些球形颗粒被池膜包裹，类似于细胞内和细胞外的包膜病毒粒子。通过蔗糖密度梯度离心纯化的突变病毒颗粒具有低感染性和转录活性，并且大多数是球形的且不含DNA。然而，颗粒制剂包含代表性的膜蛋白、裂解和未裂解的核心蛋白、病毒RNA聚合酶、早期转录因子和几种酶，这表明这些成分的掺入并不严格与DNA包装相关联。
