An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.     

An importin alpha/beta-recognized bipartite nuclear localization signal mediates targeting of the human herpes simplex virus type 1 DNA polymerase catalytic subunit pUL30 to the nucleus

Although the 1235 amino acids human herpes simplex virus type 1 (HSV-1) DNA polymerase catalytic subunit, pUL30, is essential for HSV-1 replication in the nucleus of host cells, little information is available regarding its nuclear import mechanism. The present study addresses this issue directly, characterizing pUL30's nuclear import pathway for the first time using quantitative confocal laser scanning microscopy (CLSM) on living cells, and fluorescent binding assays. In addition to a previously described nuclear localization signal (NLS) located within the pUL30 binding site for the polymerase accessory protein (PAP) pUL42, that appears to be dispensable for nuclear targeting, pUL30 possesses three putative basic NLSs. Intriguingly, the core of pUL30-NLS2 (residues 1114-1120) is highly homologous to that of the recently described NLS, similarly located upstream of the PAP binding site, of the human cytomegalovirus (HCMV) DNA polymerase catalytic subunit, pUL54. Here we show for the first time that pUL30-NLS2 itself is only partially functional in terms of nuclear import due to residue P1118 present in position 3 of the NLS core. Intriguingly, pUL30-NLS2 together with pUL30-NLS3 (residues 1133-1136) represents a fully functional bipartite NLS (pUL30-NLSbip), required for nuclear targeting of pUL30, and able to confer nuclear localization on heterologous proteins by conferring high-affinity interaction with the importin (IMP) alpha/beta heterodimer. Since nuclear targeting of HSV-1 proteins forming the replication fork is crucial for viral replication, the pUL30-NLSbip emerges for the first time as a viable therapeutic target.     

BRD7核定位信号的结构分析和功能鉴定

目的:对BRD7的核定位信号进行预测、结构分析和功能鉴定,并考察其对BRD7亚细胞定位的影响。方法:通过生物信息学对BRD7的核定位信号进行预测和结构分析,然后利用绿色荧光蛋白(GFP)介导的直接荧光和间接免疫荧光定位方法分别对核定位信号的功能进行鉴定,并考察其对BRD7亚细胞定位的影响。结果:BRD7的65～96位氨基酸残基具有潜在核定位信号(NLS)的结构特征,该核定位信号包含3簇碱性氨基酸残基,可视为由2个紧密相邻、部分重叠的双向核靶序列NLS1和NLS2组成;并发现NLS及其构成上的NLS1和NLS2均具有介导异源蛋白GFP胞核定位的功能,从而证实BRD7的65～96位残基为BRD7功能性核定位信号所在区域,且单簇碱性氨基酸残基的缺失不足以破坏其核定位信号的功能;同时发现野生型BRD7呈胞核分布,而核定位信号缺失型BRD7主要呈胞浆分布。结论:BRD7的65～96位氨基酸残基为BRD7功能性核定位信号所在区域,在BRD7胞核分布模式中发挥了十分重要的作用。     

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.     

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.     

Identification and characterization of nuclear localization signals within the nucleocapsid protein VP15 of white spot syndrome virus

The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS 1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLSI were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.