Expression, Purification, and Crystallization of Full-Length HSV-1 gB for Structure Determination

2019 ◽  
pp. 395-407
Author(s):  
Rebecca S. Cooper ◽  
Ekaterina E. Heldwein
Keyword(s):  
Structure Determination ◽  
Full Length ◽  
Hsv 1

scholarly journals Herpes Simplex Virus UL12.5 Targets Mitochondria through a Mitochondrial Localization Sequence Proximal to the N Terminus

2009 ◽  
Vol 83 (6) ◽  
pp. 2601-2610 ◽  
Author(s):  
Jennifer A. Corcoran ◽  
Holly A. Saffran ◽  
Brett A. Duguay ◽  
James R. Smiley
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Mitochondrial Genome ◽  
Nuclear Localization ◽  
Full Length ◽  
Mitochondrial Localization ◽  
Amino Terminal ◽  
The Matrix ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) gene UL12 encodes a conserved alkaline DNase with orthologues in all herpesviruses. The HSV-1 UL12 gene gives rise to two separately promoted 3′ coterminal mRNAs which encode distinct but related proteins: full-length UL12 and UL12.5, an amino-terminally truncated form that initiates at UL12 codon 127. Full-length UL12 localizes to the nucleus where it promotes the generation of mature viral genomes from larger precursors. In contrast, UL12.5 is predominantly mitochondrial and acts to trigger degradation of the mitochondrial genome early during infection. We examined the basis for these very different subcellular localization patterns. We confirmed an earlier report that the amino-terminal region of full-length UL12 is required for nuclear localization and provide evidence that multiple nuclear localization determinants are present in this region. In addition, we demonstrate that mitochondrial localization of UL12.5 relies largely on sequences located between UL12 residues 185 and 245 (UL12.5 residues 59 to 119). This region contains a sequence that resembles a typical mitochondrial matrix localization signal, and mutations that reduce the positive charge of this element severely impaired mitochondrial localization. Consistent with matrix localization, UL12.5 displayed a detergent extraction profile indistinguishable from that of the matrix protein cyclophilin D. Mitochondrial DNA depletion required the exonuclease activity of UL12.5, consistent with the idea that UL12.5 located within the matrix acts directly to destroy the mitochondrial genome. These results clarify how two highly related viral proteins are targeted to different subcellular locations with distinct functional consequences.

scholarly journals Origin of Expression of the Herpes Simplex Virus Type 1 Protein US1.5

2009 ◽  
Vol 83 (18) ◽  
pp. 9183-9194 ◽  
Author(s):  
J. Jason Bowman ◽  
Priscilla A. Schaffer
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Expression Patterns ◽  
Full Length ◽  
Early Protein ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT ICP22, an immediate-early protein of herpes simplex virus type 1 (HSV-1), is required for viral replication in nonpermissive cell types and for expression of a class of late viral proteins which includes glycoprotein C. An understanding of the mechanism of ICP22 function has been complicated by the coexpression of the full-length protein with an in-frame, C-terminus-specific protein, US1.5. In this report, we confirm that the US1.5 protein is a bona fide translation product since it is detected during infections with three laboratory strains and two low-passage clinical isolates of HSV-1. To clarify the expression patterns of the ICP22 and US1.5 proteins, we examined their synthesis from plasmids in transient expression assays. Because previous studies had identified two different US1.5 translational start sites, we attempted to determine which is correct by studying the effects of a series of deletion, nonsense, and methionine substitutions on US1.5 expression. First, amino acids 90 to 420 encoded by the ICP22 open reading frame (ORF) migrated at the mobility of US1.5 in sodium dodecyl sulfate-polyacrylamide gels. Second, introduction of a stop codon downstream of M90 ablated expression of both ICP22 and US1.5. Finally, mutation of M90 to alanine (M90A) allowed expression of full-length ICP22 while dramatically reducing expression of US1.5. Levels of US1.5 but not ICP22 protein expression were also reduced in cells infected with an M90A mutant virus. Thus, we conclude that expression of IC22 and that of US1.5 can occur independently of each other and that US1.5 translation initiates at M90 of the ICP22 ORF.

scholarly journals Expression of the HSV-1 capsid protein VP19C in Escherichia coli: A single amino acid change overcomes an expression block of the full-length polypeptide

2011 ◽  
Vol 77 (1) ◽  
pp. 80-85 ◽  
Author(s):  
Brandon W. Henson ◽  
Nicole Johnson ◽  
Alakesh Bera ◽  
Mercy E. Okoye ◽  
Keshal Viren Desai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Capsid Protein ◽  
Amino Acid Change ◽  
Full Length ◽  
Single Amino Acid ◽  
Single Amino Acid Change ◽  
Hsv 1

scholarly journals Nuclear Localizations of the Herpes Simplex Virus Type 1 Tegument Proteins VP13/14, vhs, and VP16 Precede VP22-Dependent Microtubule Reorganization and VP22 Nuclear Import

2005 ◽  
Vol 79 (8) ◽  
pp. 4730-4743 ◽  
Author(s):  
Jamie C. Yedowitz ◽  
Anna Kotsakis ◽  
Elisabeth F. M. Schlegel ◽  
John A. Blaho
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Full Length ◽  
Acetylated Tubulin ◽  
Tegument Proteins ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol. 75:8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (UL49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while α-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.

scholarly journals Full-length de novo protein structure determination from cryo-EM maps using deep learning

2020 ◽  
Author(s):  
Jiahua He ◽  
Sheng-You Huang
Keyword(s):  
Deep Learning ◽  
Structure Determination ◽  
De Novo ◽  
Protein Structures ◽  
Protein Structure Determination ◽  
Full Length ◽  
Supplementary Information ◽  
General Applicability ◽  
Data Set ◽  
Convolutional Networks

AbstractAdvances in microscopy instruments and image processing algorithms have led to an increasing number of cryo-EM maps. However, building accurate models for the EM maps at 3-5 Å resolution remains a challenging and time-consuming process. With the rapid growth of deposited EM maps, there is an increasing gap between the maps and reconstructed/modeled 3-dimensional (3D) structures. Therefore, automatic reconstruction of atomic-accuracy full-atom structures from EM maps is pressingly needed. Here, we present a semi-automatic de novo structure determination method using a deep learning-based framework, named as DeepMM, which builds atomic-accuracy all-atom models from cryo-EM maps at near-atomic resolution. In our method, the main-chain and Cα positions as well as their amino acid and secondary structure types are predicted in the EM map using Densely Connected Convolutional Networks. DeepMM was extensively validated on 40 simulated maps at 5 Å resolution and 30 experimental maps at 2.6-4.8 Å resolution as well as an EMDB-wide data set of 2931 experimental maps at 2.6-4.9 Å resolution, and compared with state-of-the-art algorithms including RosettaES, MAINMAST, and Phenix. Overall, our DeepMM algorithm obtained a significant improvement over existing methods in terms of both accuracy and coverage in building full-length protein structures on all test sets, demonstrating the efficacy and general applicability of DeepMM.Availabilityhttps://github.com/JiahuaHe/DeepMMSupplementary informationSupplementary data are available.

Resonance assignment and secondary structure determination of full length human Dickkopf 4 (hDkk4), a secreted, disulphide-rich Wnt inhibitor protein

2014 ◽  
Vol 9 (1) ◽  
pp. 147-151 ◽  
Author(s):  
Alice M. Barkell ◽  
Gill Holdsworth ◽  
Lorna C. Waters ◽  
Vaclav Veverka ◽  
Patrick M. Slocombe ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Resonance Assignment ◽  
Structure Determination ◽  
Full Length ◽  
Inhibitor Protein ◽  
Wnt Inhibitor

scholarly journals Structure Determination of Active Full Length Human Taspase1: Towards Novel Anti-Cancer Therapeutics

2019 ◽  
Vol 116 (3) ◽  
pp. 17a
Author(s):  
Jose M. Garcia ◽  
Nirupa Nagaratnam ◽  
Rebecca Jernigan ◽  
Gihan Ketawala ◽  
Silvia Delker ◽  
...  
Keyword(s):  
Structure Determination ◽  
Cancer Therapeutics ◽  
Full Length ◽  
Anti Cancer

scholarly journals Fusion on a Pedestal: The Structure of the Full-Length HSV-1 Fusogen gB

2017 ◽  
Vol 112 (3) ◽  
pp. 188a ◽  
Author(s):  
Rebecca S. Cooper ◽  
Elka R. Georgieva ◽  
Henry B. Rogalin ◽  
Peter P. Borbat ◽  
Jack H. Freed ◽  
...  
Keyword(s):  
Full Length ◽  
Hsv 1
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