Matreyek Kenneth A, Wang Weifeng, Serrao Erik, Singh Parmit Kumar, Levin Henry L, Engelman Alan
Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA, 02215, USA.
Present address: Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA.
Retrovirology. 2014 Oct 25;11:90. doi: 10.1186/s12977-014-0090-z.
Interferon-induced cellular proteins play important roles in the host response against viral infection. The Mx family of dynamin-like GTPases, which include MxA and MxB, target a wide variety of viruses. Despite considerable evidence demonstrating the breadth of antiviral activity of MxA, human MxB was only recently discovered to specifically inhibit lentiviruses. Here we assess both host and viral determinants that underlie MxB restriction of HIV-1 infection.
Heterologous expression of MxB in human osteosarcoma cells potently inhibited HIV-1 infection (~12-fold), yet had little to no effect on divergent retroviruses. The anti-HIV effect manifested as a partial block in the formation of 2-long terminal repeat circle DNA and hence nuclear import, and we accordingly found evidence for an additional post-nuclear entry block. A large number of previously characterized capsid mutations, as well as mutations that abrogated integrase activity, counteracted MxB restriction. MxB expression suppressed integration into gene-enriched regions of chromosomes, similar to affects observed previously when cells were depleted for nuclear transport factors such as transportin 3. MxB activity did not require predicted GTPase active site residues or a series of unstructured loops within the stalk domain that confer functional oligomerization to related dynamin family proteins. In contrast, we observed an N-terminal stretch of residues in MxB to harbor key determinants. Protein localization conferred by a nuclear localization signal (NLS) within the N-terminal 25 residues, which was critical, was fully rescuable by a heterologous NLS. Consistent with this observation, a heterologous nuclear export sequence (NES) abolished full-length MxB activity. We additionally mapped sub-regions within amino acids 26-90 that contribute to MxB activity, finding sequences present within residues 27-50 particularly important.
MxB inhibits HIV-1 by interfering with minimally two steps of infection, nuclear entry and post-nuclear trafficking and/or integration, without destabilizing the inherent catalytic activity of viral preintegration complexes. Putative MxB GTPase active site residues and stalk domain Loop 4 -- both previously shown to be necessary for MxA function -- were dispensable for MxB antiviral activity. Instead, we highlight subcellular localization and a yet-determined function(s) present in the unique MxB N-terminal region to be required for HIV-1 restriction.
干扰素诱导的细胞蛋白在宿主抗病毒感染反应中发挥重要作用。动力蛋白样GTP酶的Mx家族，包括Mx A和Mx B，可靶向多种病毒。尽管有大量证据表明Mx A具有广泛的抗病毒活性，但人类Mx B直到最近才被发现能特异性抑制慢病毒。在此，我们评估了Mx B限制HIV - 1感染的宿主和病毒决定因素。
在人骨肉瘤细胞中异源表达Mx B可有效抑制HIV - 1感染（约12倍），但对其他逆转录病毒几乎没有影响。抗HIV作用表现为对2 - 长末端重复序列环状DNA形成的部分阻断，进而影响核输入，因此我们发现了核后进入阻断的证据。大量先前已鉴定的衣壳突变以及消除整合酶活性的突变可抵消Mx B的限制作用。Mx B的表达抑制了病毒整合到富含基因的染色体区域，这与之前观察到的细胞中核转运因子（如运输蛋白3）缺失时的影响类似。Mx B的活性不需要预测的GTP酶活性位点残基或茎域内一系列赋予相关动力蛋白家族蛋白功能寡聚化的无结构环。相反，我们观察到Mx B N端的一段残基包含关键决定因素。由N端25个残基内的核定位信号（NLS）赋予的蛋白质定位至关重要，异源NLS可完全挽救其功能。与此观察结果一致，异源核输出序列（NES）消除了全长Mx B的活性。我们还绘制了26 - 90位氨基酸内有助于Mx B活性的子区域，发现27 - 50位残基内的序列尤为重要。
Mx B通过干扰至少两个感染步骤（核进入以及核后运输和/或整合）来抑制HIV - 1，而不会破坏病毒预整合复合物的固有催化活性。推测的Mx B GTP酶活性位点残基和茎域环4（两者先前均显示对Mx A功能是必需的）对于Mx B的抗病毒活性并非必需。相反，我们强调亚细胞定位以及独特的Mx B N端区域中尚未确定的功能对于HIV - 1限制是必需的。
