11.05.2009

1

Philipp Odermatt 12.5.2009

Inhalt• Allgemeine InformationenAllgemeine Informationen• Das Virion• Replikation• DNA Injektion

11.05.2009

2

Allgemeine Informationen• Familie: PodoviridaeFamilie: Podoviridae• ds DNA Virus (~40000 bp)• Infiziert E.Coli• Unbehüllt• Komplexe Kopf‐Schwanz Struktur• Replikationszyklus: 25 Minuten• Replikationszyklus: 25 Minuten• Pro infizierte Zelle etwa 100 Virusteilchen

Das T7 Virion

• Ikosaedrischer Kopf• Kragen• Kurzer Schwanz• 6 Schwanzfasern

11.05.2009

3

Replikation

• Die frühe Gene werden durch zelluläre RNA‐Polymerase transkribiert

• Alle übrigen Gene werden von der viralen T7 RNA‐Polymerase transkribierty

DNA Injektion• Problem: Der Schwanz des T7 

Phagen ist nicht kontraktil.• Der Schwanz des T7 Phagen ist 

zu kurz um das Zytoplasma der Wirtszelle zu erreichen.

11.05.2009

4

Injektion der T7 DNA

Durch die gleichzeitigen Signale der Schwanzfasern und des Schwanz an das Verbindungsstück (gp8) werden die irreversiblen Schritte der Infektion eingeleitet.

Injektion der T7 DNADurch Signale vom  gp 14 bildet eine Pore durch die Verbindusstück werden die Proteine gp14, gp15 und gp16 welche einen inneren Zylinder bilden freigesetzt.

äussere Membran, gp 15 und gp16 durch die Peptidoglycanschicht und die innere Membran

11.05.2009

5

gp16/gp15gp 16 hydrolysiert lokal die Peptidoglycane und bildet eine Pore durch die Pepdidoglycanschicht.

gp15 bildet eine Pore durch die innere Membran der Wirtszelle

QuellenIan J. Molineux Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712‐1095, USA; No syringes please, ejection of phage T7 DNA from the virion is enzyme drivenPhilip Serwer, Elena T Wright, Kevin W Hakala and Susan T Weintraub; Evidence for bacteriophage T7 tail extension during DNA injectionhttp://www.expasy.org/viralzone/all_by_species/518.htmlp // p y g/ / _ y_ p /5http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/02.054.0.01.001.htmBrock Mikrobiologie 11. überarbeitete Auflage; Pearson Studium

1

12.5.2009/CZ

microRNAs in SV40

Christina Zbinden

12.5.2009

12.5.2009/CZ

Contents

1) Facts: Taxonomy, Structure, Infection...

2) Molecular mechanism: microRNAs

2

12.5.2009/CZ

• unenveloped, icosahedral virion

• transmitted from monkeys to humans (contaminated polio vaccines)

• potentially oncogenic: disruption of critical cell cycle control pathways(e.g. inactivation of p53, a tumor supressor gene)

SV40 (Simian virus 40)

Simian viurs 40Species:

PolyomavirusGenus:

PolyomaviridaeFamily:

Group I (dsDNA)Group:

Simians = non-human primates

12.5.2009/CZ

5kb, circular dsDNA genome:

• Large and small tumour antigens: T ag & t ag

Tag essential for DNA replication

• 1 agnoprotein:

assembly of viral particles,processing of late mRNA

• 3 capsid proteins: VP1, VP2 & VP3

SV40 (Simian virus 40)

Late transcriptsEarly transcripts

Sullivan et al., 2005

3

12.5.2009/CZ

Infection

Late transcriptsEarly transcripts

Sullivan et al., 2005

1) Virion binds to class I MHC receptor on host cell surface by virion glycoprotein 1 (VP1)

2) Caveolae-mediated endocytosis

3) Early transcription:T ag and t ag

4) DNA replication

5) Late transcription: capsid proteins and microRNAs targeting early viralmRNAs downregulation of T ag

miRNA = endogenous small (~22nt) non-coding RNA that regulate geneexpression by targeting complementarymRNA

12.5.2009/CZ

microRNA pathway 11) Precursor molecule (pri-miRNA) is

processed by the RNAse III typeendonuclease Drosha in complexwith dsRNA-binding proteins intohairpin (pre-miRNAs)

2) Dicer cleaves hairpin into 22nt miRNA duplexes

3) One strand is selected as a maturemiRNA (other strand degraded)

in SV40 both strands becomemature miRNAs

openbiosystems.com

4

12.5.2009/CZ

microRNA pathway 2

openbiosystems.com

4) ss-miRNa is assembled intomiRNA-induced silencing complex(RISC)

5) RISC targets complementarymRNA (3´ UTR):

Imperfect complementarity:

mRNA degradation

Perfect complementarity:

Translational repression

12.5.2009/CZ

•microRNA produced by late viral mRNAtargets early viral mRNA

•Downregulation of T antigen

•Escape from detection by the host immune system:

Reduced interferon release when CTL meets SV40 infected cell

Reduced cytotoxic T lymphocyte-mediated lysis

Function of miRNAs in SV40

5

12.5.2009/CZ

Thank you for your attention!

1

Human Parechovirus

Human Parechovirus (HPeV)

Molecular Virology, FS09

Anita Walther

Taxonomy

Family: Picornaviridae

Types: six identified human viruses (I-VI)

⇒ recent studies show VIII

Closely related to Ljungan virus

Discovery: ~50 years ago

2

Epidemiology

• 60% of 580 HPeV infections in children <1 year (also neonates)

⇒ Loss of maternal antibodies in the first few months?

• Gastrointestinal, respiratory and CNS-related diseases

⇒ Encephalitis

⇒ Meningitis

⇒ Diarrhoea

⇒ Sepsis

• Transmission: faecal, oral or respiratory routes

• Types hardly to identify because of frequent recombinations in the 5’ UTR

• Spread all over the world!

Further informations: paper H. Harvala, P. Simmonds

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Molecular Biology of Human Parechovirus

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Single stranded positive sense RNA virus

non-enveloped, ~30nm in diameter

Polyprotein synthesis

Capsid:

• Protein shell surrounding the naked RNA genome

• densely-packed icosahedral arrangement

• 3 polypeptides: VP0, VP1, VP3

• No internal capsid protein

~7300b

3

The Life Cycle of typical Picornavirus

Parechovirus receptors and cell entry

Entry process:

• Immunoglobulins (Ig) and Integrin families as receptors

• Binding interaction to amino acid residues in a depression surrounding the vertices of the viral capsid (five-fold axis symmetry)

• Binding destablishes the capsid, initiation uncoating process

⇒ diversity of receptors used (maybe adaptive property of picornaviruses)

• Host cell entry through clathrin-mediated endocytotic pathway

4

Specific receptors: Integrins

• C-terminus of HPeV1 VP1 contains an arginin-glycine-aspartic acid (RGD) motif

⇒ attachment to cell surface Integrins

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Parechovirus receptors and cell entry

αVβ1 αVβ3 αVβ6-Integrins

• blocking experiments with RGD-containing motifs

Short outlook to the future

• HPeV can mutate and change its serology very quickly

• Many unpublished cases of additional types of HPeV already exist

⇒ Center of Disease Control

• Access to molecular detection and type identification methods will greatly expand the number of HPeV types in the future

⇒ genetically and antigenetically diverse as human enterovirus?

• Seroprevalence studies will provide valuable new information on emergence

• Increased surveillance is a must!