Greseth Matthew D, Czarnecki Maciej W, Bluma Matthew S, Traktman Paula
Departments of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA.
Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
J Virol. 2018 Jan 2;92(2). doi: 10.1128/JVI.01719-17. Print 2018 Jan 15.
Vaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The single-stranded DNA (ssDNA) binding protein (SSB) I3 is among the replication machinery encoded by the 195-kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus (vΔI3) lacking the I3 open reading frame (ORF). In noncomplementing CV1-CAT cells, vΔI3 shows a severe defect in the production of infectious virus (≥200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (≤0.2% of wild-type [WT] levels), and late proteins do not accumulate. When several other noncomplementing cell lines are infected with vΔI3, the yield of infectious virus is also dramatically reduced (168- to 1,776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CAT < A549 < BSC40 < HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this subgenomic DNA supports late protein expression, it does not support the production of infectious virions. Electron microscopy (EM) analysis of vΔI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vΔI3. This report provides definitive evidence that the vaccinia virus I3 protein is the replicative SSB and is essential for productive viral replication. Poxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells and necessitates that the double-stranded DNA (dsDNA) genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination, and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3 but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB.
痘苗病毒在DNA病毒中很不寻常，它仅在受感染细胞的细胞质中进行复制。单链DNA（ssDNA）结合蛋白（SSB）I3是由195kb基因组编码的复制机制的一部分，尽管对I3尚未进行直接的遗传学分析。在此，我们描述了一种互补细胞系（CV1-I3），它能完全支持缺少I3开放阅读框（ORF）的缺失病毒（vΔI3）的复制。在非互补的CV1-CAT细胞中，vΔI3在感染性病毒产生方面表现出严重缺陷（降低≥200倍）。早期蛋白质合成和核心解体正常发生。然而，DNA复制严重受损（≤野生型[WT]水平的0.2%），晚期蛋白质不积累。当其他几种非互补细胞系感染vΔI3时，感染性病毒的产量也显著降低（降低168至1776倍）。令人惊讶的是，不同细胞系（CV1-CAT < A549 < BSC40 < HeLa）中DNA积累的残留水平在1%至12%之间变化；然而，任何可检测到的新生DNA在大小上都是亚基因组的。尽管这种亚基因组DNA支持晚期蛋白质表达，但不支持感染性病毒粒子的产生。对vΔI3感染的BSC40细胞进行电子显微镜（EM）分析发现，未成熟病毒粒子大量存在，但未观察到成熟病毒粒子。相反，出现了基因组包装受阻特征的异常病毒粒子。最后，我们证明编码先前描述的具有受损ssDNA结合活性的I3变体的CV1细胞系无法互补vΔI3。本报告提供了确凿证据，表明痘苗病毒I3蛋白是复制性SSB，对病毒的有效复制至关重要。痘病毒作为感染因子、疫苗和溶瘤治疗剂，在历史和当代都具有重要意义。痘病毒在哺乳动物细胞的DNA病毒中，其细胞质复制是独特的，这使得双链DNA（dsDNA）基因组必须编码病毒复制机制。本研究聚焦于I3蛋白。作为一种ssDNA结合蛋白（SSB），尽管缺乏遗传学分析，但I3一直被认为在基因组复制、重组和修复中发挥重要作用。在此，我们报告了I3缺失病毒的特征。在没有I3表达的情况下，DNA复制严重受损，病毒产量大幅下降。在表达WT I3的细胞系中可恢复感染性病毒的产生，但在表达具有ssDNA结合活性缺陷的I3突变体的细胞系中则不能。这些数据确凿地表明I3是一种必需的病毒蛋白，其功能是病毒复制性SSB。
