Differential Phosphorylation of the Nucleoprotein of Influenza A Viruses

Kistner, Otfried; Müller, Karin; Scholtissek, C.

doi:10.1099/0022-1317-70-9-2421

Cited by 62 publications

(82 citation statements)

References 17 publications

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“…These studies reported that the drugs DRB and LB, which were added at the beginning of the infection, did not inhibit viral mRNA synthesis completely and that viral transcripts were retained in the nucleus, consistent with our microscopy results. In addition, the Protein Kinase C Inhibior [also known as 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride, kinase inhibitor H7] was found to block viral late gene expression but not the viral RNA [18,19]. Vogel et al [30] showed that the H7 resulted in retention of HA in the nuclei of infected chick embryo fibroblasts.…”

Section: Discussionmentioning

confidence: 99%

Influenza infection modulates vesicular trafficking and induces Golgi complex disruption

et al. 2016

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Influenza A virus (IFV) replicates its genome in the nucleus of infected cells and uses the cellular protein transport system for genome trafficking from the nucleus to the plasma membrane. However, many details of the mechanism of this process, and its relationship to subsequent cytoplasmic virus trafficking, have not been elucidated. We examined the effect of nuclear transport inhibitors Leptomycin B (LB), 5,6 dichloro-1-b-D-ribofuranosyl-benzimidazole (DRB), the vesicular transport inhibitor Brefeldin A (BFA), the caspase inhibitor ZWEHD, and microtubule inhibitor Nocodazole (NOC) on virus replication and intracellular trafficking of viral nucleoprotein (NP) from the nucleus to the ER and Golgi. Also, we carried out complementary studies to determine the effect of IFV on intracellular membranes. Inhibition of the CRM1 and TAP-P15 nuclear transport pathways by DRB and LB blocked completely the export of virus. Inhibition of vesicular trafficking by BFA, NOC, and ZWEHD also affected influenza infection. Interestingly, IFV infection induced fragmentation of the Golgi complex resulting in diffuse distribution of large and small vesicles throughout the cytoplasm. Live-cell microscopy revealed expansion of Golgi localization signals indicating progressive dispersion of Golgi positive structures, resulting in the disassembly of the Golgi ribbon structure. Other vesicular components (Rab1b, ARF1 and GBF1) were also found to be required for IFV infection. Furthermore, the exact step at which IFV infection disrupts vesicle trafficking was identified as the ER-Golgi intermediate compartment. These findings suggest that IFV NP is trafficked from the nucleus via the CRM1 and TAP pathways. IFV modulates vesicular trafficking inducing disruption of the Golgi complex. These studies provide insight on the ways in which IFV affects intracellular trafficking of different host proteins and will facilitate identification of useful pharmaceutical targets to abrogate virus replication.

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Section: Discussionmentioning

confidence: 99%

Influenza infection modulates vesicular trafficking and induces Golgi complex disruption

et al. 2016

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“…Immunoprecipitations were carried out as described 6 with a polyclonal rabbit antiserum against the N terminus of HA, when indicated (NHA), or with a polyclonal rabbit antiserum (P) against X-31 virus, which recognizes all forms of HA as well as the nucleoprotein (NP), which is a phosphoprotein [32][33][34] and can appear as either one or two bands in the gel depending on the phosphorylation status of the protein (A. Ora, Utrecht University, and I.B., unpublished observations). To probe for different conformations, two mouse monoclonal IgG antibodies, F1 and F2, made against nondenatured HA2 were used 6 .…”

Section: Immunoprecipitation and Sds-pagementioning

confidence: 99%

A critical step in the folding of influenza virus HA determined with a novel folding assay

Maggioni

Liscaljet

Braakman

2005

Nat Struct Mol Biol

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Most principles of protein folding emerged from refolding studies in vitro on small, soluble proteins, because large ones tend to misfold and aggregate. We developed a folding assay allowing the study of large proteins in detergent such that the extent of cellular assistance required for proper folding can be determined. We identified a critical step in the in vivo folding pathway of influenza virus hemagglutinin (HA). Only the formation of the first few disulfides in the top domain of HA required the intact endoplasmic reticulum. After that, HA proceeded to fold efficiently in a very dilute solution, despite its size and complexity. This study paves the way for detailed structural analyses during the folding of complex proteins.Protein folding is the process by which linear polypeptide chains acquire their biologically active three-dimensional conformation. Although all information needed to reach the native conformation is encoded by the amino acid sequence 1 , protein folding inside the cell generally is assisted by molecular chaperones and folding enzymes 2-4 . They mainly protect nascent chains and newly synthesized proteins from misfolding and aggregation. Chaperones and folding enzymes reside in almost every compartment of eukaryotic cells.The endoplasmic reticulum (ER) is highly specialized for the folding of membrane and secretory proteins. One of the bestcharacterized model proteins used to study protein folding in the ER is the influenza virus HA. This protein is not only crucial for influenza virus infection but also an important target for the immune system. HA is a multidomain glycoprotein of 84 kDa with six disulfide bonds and seven N-linked glycans. It folds in the ER, trimerizes and is transported to the plasma membrane, where it is incorporated into new virions. The folding of HA has been studied extensively both in complete cells [5][6][7][8][9][10][11] and in microsomes 12,13 , using radioactive labeling of newly synthesized HA or truncated ribosome-bound nascent chains 14 . Although these studies yielded fairly detailed information on the HA folding process, further molecular insight would require biophysical studies on purified protein, which would disregard the role of the cell.To start bridging the gap between folding studies in vitro and in intact cells, we developed an assay that allows the study of post-translational HA folding in a cell-free system without membranes. We replaced the chase period in the well-established pulse-chase folding assay 6 with a folding analysis in solution: the protein is translated and translocated in intact cells, after which post-translational folding is studied in a detergent cell lysate. Using this 'in vitro chase' assay, we found that HA can fold in a detergent cell lysate without ER assistance, provided that the first folding step occurs in an intact ER. RESULTS HA can fold in a detergent cell lysateWhen studied with the pulse-chase assay in intact cells, newly synthesized HA ran as a single band in a reducing SDS-PAGE gel (Fig. 1a, R) and as three ban...

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“…Restriction is said to be partial because of the evidence for occurrences of interspecies virus transmission, e.g., the finding of avian HlNl viruses in swine in Europe, human H3N2 viruses in swine, swine HlNi viruses in humans, and other avian viruses in seals, whales, and mink. (88,89,148). Also, avian NP specifically causes restricted replication of otherwise human viruses in squirrel monkeys (162).…”

Section: Molecular Determinants Of Host Range Restrictionmentioning

confidence: 99%