Viral Vectors

May 8

Viral vectors

• Herpes simplex virus amplicons

• Adenovirus vectors

• Lentivirus vectors

Herpes simplex virus amplicons

HSV replicative cycle

IE genesE genes

L genesDNA replication

viral DNA

gB

gC

gD

vhs

tif

inclusion body cell-cellspreadsuppression of

class I MHC

HSV amplicons

promoter pA pack

HS

V-o

ri

bacterial plasmid seq

cloning site defectivevirus

cosmidsHSV genes

BACSHSV geneslinear plasmid joined end to end

150kb packaged into virus particle

HSV ampliconsadvantages:

• large packaging capacity

• for small inserts - large gene dose

• extra-chromosomal

• wide host range

• no viral sequences except ori and pkg

HSV ampliconsdisadvantages

• often low virus titres

• short-term expression due to silencing

Improving expression by amplicons

Plasmid DNA Sequences Present in Conventional Herpes Simplex Virus Amplicon Vectors Cause Rapid Transgene Silencing by Forming Inactive Chromatin Masataka Suzuki, Kazue Kasai, and Yoshinaga Saeki, J. Virol 80: 3293, April 2006

Adenovirus vectors

• gene of interest part of defective virus genome

• exrta-chromosomal

Clontech Adeno-X expression system

Lentivirus expression systems

• contain no genes for viral structural genes

• wide host range if VSV-G used

• integrate into host chromosome (dividing or stationary cells)

A retrovirus virionenvelope (ENV)

nucleocapsidp27 (GAG)

genome (2 copies)

RNA dep. DNA pol(POL)

p15e

gp70

The viral genome (oncornaviruses)

LTR LTRGAG POL ENV

Long terminalrepeat

Group specific antigens(nucleocapsid)

RNA dependant DNApolymerase

Envelope proteins(type-specific antigens)

All three genes - GAG, POL, ENV - required for replication

Replicative cyclereceptor

uncoating

RNA

ds DNA

proteinsmRNA

viral dsDNA integratedinto host genome

genomic RNA

blockedreceptors

assemblypro-virus

Directional cloning into pLenti

Invitrogen Lentivirus vector systemclone gene of interest

plasmids expressing:HIV Gag and PolHIV RevVSV G

cells

lentivirus virions with gene of interest

into target cells

stably expressingcells(blasticidin)

transient expression

Primers for amplifying ZFsequence at 5’ end of ZF

5’ ATG GAC TAC AAA GAC GAT GAC GAC AAG CAA TTC ATG GAG GAG GAG GCG ATC GCC AGC CTC CCG GGG GAA GAG…

FLAG

Upstream primersFLAG+5’ CACC ATG GAC TAC AAA GAC GAT G

FLAG-5’ CACC ATG GAG GAG GAG GCG ATC G

Primers for amplifying ZFSequence of 3’ end of cds of hZhangfei

5’ GCG TCG TCT TCT CTT AAA ATG TAG 3’ CGC AGC AGA AGA GAA TTT TAC ATC

Down stream primer:5’ CAT TTT AAG AGA AGA CGA CGC 3’

Stop codon removed and sequence in phase with SV5 epitopeNo CACC at 5’ end