Zhao Xin, Tikoo Suresh K
VIDO-InterVac, University of Saskatchewan, Saskatoon, SK, Canada.
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK, Canada.
Front Microbiol. 2021 Jan 6;11:579593. doi: 10.3389/fmicb.2020.579593. eCollection 2020.
The L2 region of bovine adenovirus-3 (BAdV-3) encodes a genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81-120, 190-210, and 380-389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/β nuclear import receptor pathway. Moreover, pV amino acids 21-50 and 380-389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380-389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.
牛腺病毒3型（BAdV - 3）的L2区域编码一种属特异性蛋白，命名为pV，它对于子代病毒的产生很重要。在此，我们证明以55 kDa蛋白形式表达的BAdV - 3 pV定位于细胞核，并特异性靶向感染细胞的核仁。对pV缺失突变体的分析表明，氨基酸81 - 120、190 - 210和380 - 389作为多个核定位信号（NLS），它们似乎也作为输入蛋白α - 3蛋白（输入蛋白α/β核输入受体途径的成员）的结合位点。此外，pV氨基酸21 - 50和380 - 389似乎作为核仁定位信号（NoLs）。有趣的是，氨基酸380 - 389似乎既作为NLS又作为NoLS发挥作用。NoLs的存在对于感染性子代病毒粒子的产生至关重要，因为两个NoLs的缺失对于感染性BAdV - 3的产生是致命的。对在非互补CRL细胞中含有两个NoLs缺失/突变的突变体BAV.pVd1d3（在pV互补CRL细胞中分离得到）的分析不仅揭示了突变体pV细胞内定位改变，还降低了一些晚期蛋白的表达。然而，它似乎不影响包括突变体pV在内的病毒蛋白在BAV.pVd1d3病毒粒子中的掺入。对氯化铯纯化的BAV.pVd1d3的进一步分析表明存在衣壳被破坏的热不稳定病毒粒子，这似乎影响子代病毒粒子的感染性。我们的结果表明pV包含重叠和非重叠的NoLS/NLS。此外，两个NoLs的存在对于稳定且感染性的子代BAV.pVd1d3病毒粒子的产生似乎至关重要。
