Herpes simplex virus type 1 nuclear targeting is mediated by dynein and dynactin, but does not require the small capsid protein VP26 [Elektronische Ressource] / von Katinka Döhner

Katinka Döhner

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2006
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 25 Mo

Extrait

Herpes simplex virus type 1 nuclear targeting is
mediated by dynein and dynactin, but does not
require the small capsid protein VP26


Von der Naturwissenschaftlichen Fakultät der Universität Hannover
zur Erlangung des Grades einer

Doktorin der Naturwissenschaften
Dr. rer. nat.

genehmigte Dissertation
von


Diplom-Biochemikerin Katinka Döhner
geboren am 21. Oktober 1974 in Leverkusen

2006














Referentin: HD PD Dr. Beate Sodeik
Korreferent: Prof. Dr. Ernst Ungewickell

Tag der Promotion: 17. Mai 2006 Table of Contents 1



List of Abbreviations ........................................................................................................................ 2
1 Abstract ........................................................................................................................................... 3
2 Zusammenfassung .......................................................................................................................... 4
3 Introduction..................................................................................................................................... 6
3.1 Intracellular trafficking of viral particles ................................................................................... 6
3.2 The cytoskeleton and motor proteins .........................................................................................6
3.2.1 Intermediate filaments...................................................................................................... 6
3.2.2 Actin filaments................................................................................................................. 7
3.2.3 Myosins catalyze transport along actin filaments............................................................. 7
3.2.4 Microtubules are the highways for long distance transport.............................................. 8
3.2.5 Cytoplasmic dynein and its cofactor dynactin.................................................................. 9
3.2.6 The kinesin superfamily ................................................................................................. 12
3.3 Herpesviridae ........................................................................................................................... 14
3.4 Herpes simplex virus type 1 ..................................................................................................... 15
3.4.1 HSV1 entry into cells ..................................................................................................... 18
3.4.2 HSV1 cytosolic transport during entry........................................................................... 20
3.4.3 Potential role of microtubules during assembly and egress of HSV1 ............................ 21
3.4.4 Potential receptors for dynein, dynactin and kinesins on the HSV1 virion.................... 24
3.5 Life cell imaging of alphaherpesvirinae................................................................................... 28
3.6 Aim of the study....................................................................................................................... 30
4 Discussion ...................................................................................................................................... 31
4.1 MTs are required for efficient nuclear targeting of Ad and HSV1 .......................................... 31
4.2 Function of dynein and dynactin in HSV1 capsid transport..................................................... 33
4.3 Bidirectional HSV1 transport along MTs ................................................................................ 35
4.4 High quality virus preparations are required to analyze cell entry of HSV1 ........................... 37
4.5 The formation of hexon-specific VP5 epitopes requires VP26................................................ 38
4.6 HSV1- ∆VP26 and HSV1-GFPVP26 require dynein/dynactin for efficient nuclear targeting. 38
5 Conclusions.................................................................................................................................... 42
6 Outlook .......................................................................................................................................... 44
7 Acknowledgements ....................................................................................................................... 48
8 References...................................................................................................................................... 50
9 Appendix....................................................................................................................... 62
9.1 Curriculum vitae....................................................................................................................... 62
9.2 Publications............... 66 List of Abbreviations 2


AAA ATPase associated with various cellular activities
Ad adenovirus
ADP adenosine diphosphate
AMP-PNP adenosine 5´-( β, γ-imido)triphosphate
Arp actin-related protein
ATP triphosphate
BAC bacterial artificial chromosome
cGMP cyclic guanosine monophosphate
CGN cis-Golgi network
CLIP-170 cytoplasmic linker protein 170
DHC dynein heavy chain
DIC dyintermediate chain
DLC ylight chain
DLIC nein liintermediate chain
DNA deoxyribonucleic acid
EBV Epstein-Barr virus
EHNA erythro-9-3-[2-hydroxynonyl]adenine
ER endoplasmic reticulum
GFP green fluorescent protein
GST glutathione-S-transferase
gX glycoprotein X
HCMV human cytomegalovirus
HHV herpesvirus
HIV an immunodeficiency virus
HSV1 herpes simplex virus type 1
HSV2 pype 2
HVEM herpes virus entry mediator
ICP infected cell protein
KAP kinesin-associated
KHC kinesin heavy chain
KLC light
KSHV Kaposi’s sarcoma-associated herpesvirus
ND10 nuclear domain 10
NPC pore complex
MOI multiplicity of infection
mRFP onomeric red fluorescent protein
mRNA messenger ribonucleic acid
MT microtubule
MTOC organizing center
PAT1 protein interacting with amyloid precursor protein tail 1
PCR polymerase chain reaction
PFU plaque forming units
PRV pseudorabies virus
RNA ribonucleic acid
TGN trans-Golgi network
UL unique long
US short
VP virion protein
VV vaccinia virus
VZV varicella zoster virus
wt wildtype Abstract 3


1 Abstract
Herpes simplex virus type 1 (HSV1) infects keratinocytes and epithelial cells of the oral and perioral
region. Amplified virus enters neurons innervating that area and is transported retrogradely to the
neuronal nuclei located in the cranial ganglia, where it can establish a latent infection. After
reactivation, progeny virus is transported anterogradely to the synapse. After release from the synapse
it can reinfect the epithelium and cause recurrent disease. Thus, during several stages of its life cycle,
the neurotropic alphaherpesvirus HSV1 depends on intracellular long distance transport.
After fusion of the viral envelope and a cellular membrane, incoming capsids are transported along
microtubules (MTs) to the MT-organizing center (MTOC) and further to the nucleus. At the nuclear
pore they release their genome into the nucleoplasm, where viral replication, transcription and capsid
assembly take place.
During my Ph.D. thesis I was involved in a project which confirmed with quantitative assays that
MTs are required for efficient nuclear targeting of HSV1. Incoming HSV1 capsids colocalized with
the minus-end directed MT motor cytoplasmic dynein and its cofactor dynactin. Interfering with
dynein function by overexpressing the dynactin subunit dynamitin reduced nuclear targeting of HSV1,
and as a consequence expression of immediate-early viral genes, while virus binding and virus
internalization were not affected, and only slight alterations of the MT network were observed. Thus,
dynein and its cofactor dynactin mediate nuclear targeting of HSV1. In some cells overexpressing
dynamitin HSV1 particles accumulated in the cell periphery, and this peripheral accumulation required
MTs, suggesting that it was mediated by a plus-end directed MT motor of the kinesin family.
The small capsid protein VP26 can interact with the 14 kDa dynein light chains Tctex-1 and rp3.
Therefore, we analyzed the cell entry of HSV1- ∆VP26, which lacks VP26, and HSV1-GFPVP26,
which contains GFPVP26 instead of VP26. Both required MTs and functional dynactin for efficient
nuclear targeting in Vero and PtK cells, and consequently for efficient expression of immediate-early 2
viral genes. Since Vero cells are infected after fusion at the plasma membrane, we conclude that
cytosolic capsids lacking the potential dynein receptor VP26 can use MTs, dynein and dynactin for
efficient nuclear targeting. Moreover, capsids from HSV1-wildtype, HSV1- ∆VP26 and HSV1-
GFPVP26 bound to dynein and dynactin in vitro with similar efficiency. Compared to nuclear capsids
with almost no tegument and capsids wit