scholarly journals Autographa californica Nucleopolyhedrovirusac75is Required for the Nuclear Egress of Nucleocapsids and Intranuclear Microvesicle Formation

2017 ◽  
pp. JVI.01509-17 ◽  
Author(s):  
Anqi Shi ◽  
Zhaoyang Hu ◽  
Yachao Zuo ◽  
Yan Wang ◽  
Wenbi Wu ◽  
...  
Keyword(s):  
Life Cycle ◽  
Membrane Protein ◽  
Nuclear Membrane ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Autographa Californica ◽  
Bimolecular Fluorescence Complementation ◽  
Nuclear Egress ◽  
Ring Zone ◽  
Virion Morphogenesis

Autographa californica nucleopolyhedrovirus (AcMNPV)orf75(ac75) is a highly conserved gene of unknown function. In this study, we constructed anac75knockout AcMNPV bacmid and investigated the role ofac75in the baculovirus life cycle. The expression and distribution of the Ac75 protein were characterized, and its interaction with another viral protein was analyzed to further understand its function. Our data indicated thatac75was required for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent budded virion (BV) formation, as well as occlusion-derived virion (ODV) envelopment and embedding of ODVs into polyhedra. Western blot analyses showed that two forms of 18 and 15 kDa of FLAG-tagged Ac75 protein were detected. Ac75 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV; the 18-kDa form was associated with only BVs, whereas the 15-kDa form was associated with both types of virion. Ac75 was predominantly localized in the intranuclear ring zone during infection and exhibited a nuclear rim distribution during the early phase of infection. A phase-separation assay suggested that Ac75 was not an integral membrane protein. A coimmunoprecipitation assay revealed an interaction between Ac75 and the integral membrane protein Ac76, and bimolecular fluorescence complementation assays identified the sites of the interaction within the cytoplasm, at the nuclear membrane and ring zone in AcMNPV-infected cells. Our results have identifiedac75as a second gene that is required for both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles.IMPORTANCEDuring the baculovirus life cycle, the morphogenesis of both budded virions (BVs) and occlusion-derived virions (ODVs) is proposed to involve a budding process at the nuclear membrane, which occurs while nucleocapsids egress from the nucleus or when intranuclear microvesicles are produced. However, the exact mechanism of virion morphogenesis remains unknown. In this study, we identifiedac75as a second gene, in addition toac93, that is essential for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. Ac75 is not an integral membrane protein. However, it interacts with an integral membrane protein (Ac76) and is associated with the nuclear membrane. These data enhance our understanding of the commonalities between nuclear egress of nucleocapsids and intranuclear microvesicle formation and may help to reveal insights into the mechanism of baculovirus virion morphogenesis.

scholarly journals A Non-Conserved p33 Protein of Citrus Tristeza Virus Interacts with Multiple Viral Partners

2020 ◽  
Vol 33 (6) ◽  
pp. 859-870 ◽  
Author(s):  
Thi Nguyet Minh Dao ◽  
Sung-Hwan Kang ◽  
Aurélie Bak ◽  
Svetlana Y. Folimonova
Keyword(s):  
Host Range ◽  
Citrus Tristeza Virus ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Open Reading Frames ◽  
Bimolecular Fluorescence Complementation ◽  
In Planta ◽  
Tristeza Virus ◽  
Citrus Tristeza

The RNA genome of citrus tristeza virus (CTV), one of the most damaging viral pathogens of citrus, contains 12 open reading frames resulting in production of at least 19 proteins. Previous studies on the intraviral interactome of CTV revealed self-interaction of the viral RNA-dependent RNA polymerase, the major coat protein (CP), p20, p23, and p33 proteins, while heterologous interactions between the CTV proteins have not been characterized. In this work, we examined interactions between the p33 protein, a nonconserved protein of CTV, which performs multiple functions in the virus infection cycle and is needed for virus ability to infect the extended host range, with other CTV proteins shown to mediate virus interactions with its plant hosts. Using yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays, we demonstrated that p33 interacts with three viral proteins, i.e., CP, p20, and p23, in vivo and in planta. Coexpression of p33, which is an integral membrane protein, resulted in a shift in the localization of the p20 and p23 proteins toward the subcellular crude-membrane fraction. Upon CTV infection, the four proteins colocalized in the CTV replication factories. In addition, three of them, CP, p20, and p23, were found in the p33-formed membranous structures. Using bioinformatic analyses and mutagenesis, we found that the N-terminus of p33 is involved in the interactions with all three protein partners. A potential role of these interactions in virus ability to infect the extended host range is discussed.

scholarly journals Backbone structure of a small helical integral membrane protein: A unique structural characterization

2008 ◽  
pp. NA-NA ◽  
Author(s):  
Richard C. Page ◽  
Sangwon Lee ◽  
Jacob D. Moore ◽  
Stanley J. Opella ◽  
Timothy A. Cross
Keyword(s):  
Membrane Protein ◽  
Structural Characterization ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Backbone Structure

The E3-11.6K protein of adenovirus is an asn-glycosylated integral membrane protein that localizes to the nuclear membrane

Virology ◽  
1992 ◽  
Vol 191 (2) ◽  
pp. 743-753 ◽  
Author(s):  
Abraham Scaria ◽  
Ann E. Tollefson ◽  
Sankar K. Saha ◽  
William S.M. Wold
Keyword(s):  
Membrane Protein ◽  
Nuclear Membrane ◽  
Integral Membrane Protein

scholarly journals A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis

2021 ◽  
Author(s):  
Elmar Schiebel ◽  
Wanlu Zhang ◽  
Azqa Khan ◽  
Jlenia Vitale ◽  
Annett Neuner ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Phosphatidic Acid ◽  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Integral Membrane Protein ◽  
Nuclear Pore ◽  
Binding Properties ◽  
Perinuclear Space ◽  
Α Helix

The integral membrane protein Apq12 is an important nuclear envelope (NE)/ER modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting upon Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE during NPC biogenesis.

scholarly journals Host Vesicle Fusion Proteins VAPB, Rab11b and Rab18 Contribute to HSV-1 Infectivity by Facilitating Egress through the Nuclear Membrane

2016 ◽  
Author(s):  
Natalia Saiz-Ros ◽  
Rafal Czapiewski ◽  
Andrew Stevenson ◽  
Ilaria Epifano ◽  
Selene K. Swanson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Life Cycle ◽  
Nuclear Envelope ◽  
Host Cell ◽  
Nuclear Membrane ◽  
Fusion Proteins ◽  
Vesicle Fusion ◽  
Endoplasmic Reticulum Membrane ◽  
Nuclear Egress ◽  
Hsv 1

AbstractThe herpesvirus process of primary envelopment and de-envelopment as viral particles exit the nucleus has been for many years one of the least understood steps in the virus life cycle. Though viral proteins such as pUL31, pUL34, pUS3 and others are clearly important, these are likely insufficient for efficient fusion with the nuclear membrane. We postulated that host nuclear membrane proteins involved in virus nuclear egress would move from the inner to outer nuclear membranes due to membrane fusion events in primary envelopment and de-envelopment and then diffuse into the endoplasmic reticulum. Membrane fractions were prepared enriched in the nuclear envelope or the endoplasmic reticulum with and without HSV-1 infection and analyzed by mass spectrometry, revealing several vesicle fusion proteins as candidates in the viral nuclear egress pathway. Knockdown of three of these, VAPB, Rab11b, and Rab18, significantly reduced titers of released virus while yielding nuclear accumulation of encapsidated particles. Antibody staining revealed that VAPB visually accumulates in the inner nuclear membrane during HSV-1 infection. VAPB also co-localizes at early time points with the viral pUL34 protein known to be involved in nuclear egress. Most strikingly, VAPB was also observed on HSV-1 virus particles by immunogold labelling electron microscopy. Thus, these data reveal several new host cell vesicle fusion proteins involved in viral nuclear egress.Author SummaryHuman herpesviruses are associated with common human diseases such as chicken pox, shingles and mononucleosis and infect a wide range of animals making them economically important pathogens for livestock. Herpes simplex virus 1 (HSV-1) is most commonly associated with cold sores, but is also the leading cause of blindness by infection in the Western world. All herpesviruses share many aspects of infection. As large nuclear replicating dsDNA viruses with capsid sizes too large to use the nuclear pores to exit the nucleus, they have evolved a complex mechanism for envelopment and de-envelopment of primary herpesvirus particles, but this critical step in the virus lifecycle remains poorly understood. We have identified several host cell vesicle fusion proteins, VAPB, Rab11b and Rab18 that appear to contribute to this step in the HSV-1 life cycle. VAPB accumulates at the nuclear envelope with the HSV-1 pUL34 protein important for viral nuclear egress. Knockdown of any of these vesicle fusion proteins reduces viral titers, further arguing that they are important for nuclear egress. As there appears to be a specific subset of vesicle fusion proteins involved in viral egress, they could possibly represent novel targets for therapeutic interventions.

THE THREE LIVES OF M13 COAT PROTEIN: A VIRION CAPSID, AN INTEGRAL MEMBRANE PROTEIN, AND A SOLUBLE CYTOPLASMIC PROPROTEIN

1980 ◽  
Vol 343 (1 Precursor Pro) ◽  
pp. 384-390 ◽  
Author(s):  
William Wickner ◽  
Koreaki Ito ◽  
Gail Mandel ◽  
Marjorie Bates ◽  
Marja Nokelainen ◽  
...  
Keyword(s):  
Coat Protein ◽  
Membrane Protein ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Three Lives

scholarly journals Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

1992 ◽  
Vol 12 (7) ◽  
pp. 3288-3296 ◽  
Author(s):  
D Feldheim ◽  
J Rothblatt ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Carboxyl Terminus ◽  
Endoplasmic Reticulum Membrane ◽  
Cell Fractionation ◽  
Network Cell

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

scholarly journals SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex.

1992 ◽  
Vol 119 (3) ◽  
pp. 513-521 ◽  
Author(s):  
K G Hardwick ◽  
H R Pelham
Keyword(s):  
Membrane Protein ◽  
Golgi Complex ◽  
Vesicular Transport ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Carboxypeptidase Y ◽  
Hydrophobic Residues ◽  
Golgi Transport ◽  
Cytoplasmic Face ◽  
Multiple Copies

The ERD2 gene, which encodes the yeast HDEL (His-Asp-Glu-Leu) receptor, is essential for growth (Semenza, J. C., K. G. Hardwick, N. Dean, and H. R. B. Pelham. 1990. Cell. 61:1349-1357; Lewis, M. J., D. J. Sweet, and H. R. B. Pelham. 1990. Cell. 61:1359-1363). SED5, when present in multiple copies, enables cells to grow in the absence of Erd2p. Sequence analysis of SED5 reveals no significant homology with ERD2 or other known genes. We have raised antibodies to Sed5p which specifically recognize a 39-kD integral membrane protein. A stretch of hydrophobic residues at the COOH terminus is predicted to hold Sed5p on the cytoplasmic face of intracellular membranes. Cells that are depleted of Sed5p are unable to transport carboxypeptidase Y to the Golgi complex, and stop growing after a dramatic accumulation of ER membranes and vesicles. We conclude that the SED5 gene is essential for growth and that Sed5p is required for ER to Golgi transport. When Sed5p is overexpressed the efficiency of ER to Golgi transport is reduced, vesicles accumulate, and cellular morphology is perturbed. Immunofluorescence studies reveal that the bulk of Sed5p is not found on ER membranes but on punctate structures throughout the cytoplasm, the number of which increases upon SED5 overexpression. We suggest that Sed5p has an essential role in vesicular transport between ER and Golgi compartments and that it may itself cycle between these organelles.

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation

1992 ◽  
Vol 12 (7) ◽  
pp. 3288-3296
Author(s):  
D Feldheim ◽  
J Rothblatt ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Carboxyl Terminus ◽  
Endoplasmic Reticulum Membrane ◽  
Cell Fractionation ◽  
Network Cell

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

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