Transcript Replication of Small DNA Virus

Replication of
Small DNA
Virus
Animal Virus DNA Genomes
• Larger size range (5 – 200 kbp) than RNA
viruses
• Similar to host genome, may use cell
machinery for DNA replication &
transcription
• Problems of dependence on host cell for
DNA replication:
– Cell must be in S phase for DNA synthesis
– DNA synthesis can’t occur at ends of linear
DNA molecules (“end problem” due to use of
RNA primer)
Virus Solutions For Cell
Cycle S Phase Requirement
• Small DNA viruses only infect cells that
have entered the S phase of the cell cycle
• Other viruses induce its host cell to enter
S phase
• Large viruses encode their own enzymes
and thus not dependent on cell enzymes
Virus Solutions To DNA
Synthesis “End Problem”
• Use protein primers instead of host
cell RNA primer
• Circular genome form concatemers
(rolling circle model) with no ends
• May resort to reverse transcription
(host cell telomerase), RNA to DNA
Family Papovaviridae
• Two groups of similar viruses of animals
and humans
• Papillomavirus
• Polyoma virus, vacuolating virus
• Circular dsDNA
• Icosahedral capsid, 45-55 nm
• Replication occurs in nucleus
Genus: Papilloma Virus
• “nipple-shaped” projection, tumor
• Human papillomaviruses (HPV) 1-48
• Benign warts and tumors
(oral/pharyngeal, skin)
• Malignant tumors (oral/pharyngeal,
genital carcinoma)
• Difficult to culture in vitro
Genus: Polyoma Virus
• “many tumors” in experimental animals
• SV40 (simian vacuolating virus) - isolated
in 1960 from monkey
• JC virus – isolated in 1970 from patient
with neurologic disease
• BK virus – isolated in 1971 from kidney
transplant patient
Virus Infections
• Asymptomatic – no disease symptoms
• Acute – disease symptoms
• Persistent – long term
– Chronic: infectious virus
– Latent: no virus replication, virus reactivation
• Transformation – alter cell regulation,
tumor production
– No infectious virus
– Viral DNA, complete or partial
SV40: Host / Infections
• Primary monkey kidney cell culture =
Latent infection
• Many experimental animals (host cell lacks
some requirement for virus replication) =
Nonpermissive (“abortive”) infection
• Newborn hamsters = no virus replication,
instead Transform cells to many different
tumors
SV40: Type of Infection
Due to Host
• Monkey: Acute productive – infectious
virus
• Monkey: Persistent chronic – low level of
virus replication over long term
• Monkey: Persistent latent – no virus
replication, possible later reactivation of
virus
• Hamster: Nonproductive – no virus
replication; may lead to transformation by
disrupting cell regulation; complete or part
of viral DNA present
JC Virus Infection
• Patient with rare fatal neurologic disease –
PML (progressive multifocal
leukoencephalopathy)
• Common infection in young children via
respiratory route
• Persistent latent infection (no infectious
virus) of lungs & kidneys
• Host immunosuppression leads to
activation of virus that spreads to brain
BK Virus Infection
• Isolated from urine of renal transplant
patient
• Common mild respiratory tract infection
• Persistent latent infection of lungs and
kidney
• Host immunosuppression leads to
activation of virus
• Overt disease is rare
Papovavirus Genome:
ds DNA
• Circular, supercoiled,
condensed by cell histones
• Papillomavirus, 8 kb; may exist
as episome in host cell
• Polyoma virus, 5 kb; may
integrate into host cell DNA
• Promoter, enhancer regions
• “early” and “late” genes
Virus Infection: Cell
Transformation
SV40 Genome: Control
Region
• ori – origin of
replication for DNA
• PE, PL – promoter region
for “early” and “late”
mRNA
• “21”bp & “72”bp –
“early” promoter
enhancer regions
• T – three different
binding sites for large T
(tumor) antigen
SV40: Entry / Uncoating
• Receptor mediated endocytosis
• Transport of vesicle to nucleus
• Fusion of vesicle with nuclear
membrane and virus enters nucleus
• Uncoat and release of viral DNA in
nucleus
SV40: “Early” mRNA Transcription
• Uses #1 DNA strand
(counter-clockwise)
• “early” promoter (PE)
directs mRNA
transcription
• “TATA” box –
conscensus sequence for
cell RNA pol II
• Viral enhancer region
for cell DNA binding
proteins
• One “early” mRNA
• Alternative splicing for
two mRNA
SV40: “Early” Proteins
• Large T antigen (multifunctional):
– Activate host cell (bind and inactivate cell
growth-suppressor proteins: p105 Rb, p53)
– Block cell apoptosis (programmed death)
– Viral DNA replication
– Down-regulate “early” mRNA
– Activate “late” mRNA
– Role in virus assembly
• Small t antigen (viral DNA replication)
Tumor (Cellular Growth)
Suppressor Genes: Rb, p53
• Cellular genes whose function is to block
uncontrolled cell replication
• Rb (retinoblastoma susceptibility gene):
– Gene product (p105 RB) repress transcription
– Mutation results in tumor (uncontolled cell
growth) of retina
• p53 gene product (p53) leads to:
– G1 arrest; contact-inhibition
– Apoptosis (programmed cell death)
– Inactivation of p53 results in loss of cell
division repression
SV40: Semi-conservative
DNA Replication
• Formation of initiation complex:
– T antigen (numerous enzymes)
– Cell DNA primase-DNA pol
– Cell DNA binding proteins
• dsDNA opens up, RNA primer
• Bidirectional DNA (continous, discontinous
strand) synthesis
• Forms two dsDNA “loops”
SV40: DNA Replication
• dsDNA opens up,
RNA primer
• Bidirectional DNA
(continous,
discontinous
strand) synthesis
• Forms two dsDNA
“loops”
SV40: “Late” mRNA Transcription
• Follows viral DNA
replication
• Uses #2 DNA strand
(clockwise)
• T antigen binds near Ori
and blocks “early” mRNA
transcription
• T antigen binds to
Enhancer region and
turns-on “late” promoter
(PL)
• One “late” mRNA
• Alternative splicing for
two mRNAs
SV40: “Late” Proteins
• Two “late” mRNAs (both
are bicistronic)
• Each mRNA translates
for two proteins using
different start codons:
– #1 mRNA: VP 1,
agnoprotein
– #2 mRNA: VP2, VP3
SV40:
Maturation
• Assembly of
virus in nucleus
• Release by cell
lysis
• In cell culture,
produce ~ 104 105 progeny
virus
Oncogenes
• Genes encoding the proteins originally
identified as the transforming agents of
oncogenic viruses (SV40; T antigen)
• Some oncogenes were shown to be normal
components of cells
• For retrovirus:
– v-onc is viral oncogene
– c-onc is cellular version and termed protooncogene and are cellular growth control genes
– Most likely v-onc “stolen” from host cell
Nonpermissive SV40 Infection
• “abortive” infection as
viral DNA can not
replicate in host cell
• Viral T antigen
stimulates cell DNA
replication and cell
division; continued
stimulation may lead to
cell transformation
• Viral DNA may integrate
into cell DNA by random
recombination event,
results in stable cell
transformation
pSV40T7 Recombinant
Expression Vector
• SV40 signals for mRNA transcription of
cDNA cloned into polycloning site
– SV40 “early” promoter (PE)
– SV40 polyadenylation site
Reading & Questions
• Chapter 16: Replication Strategies
of Small and Medium-Sized DNA
Viruses
Class Discussion –
Lecture 9
• 1. Why does SV40 require its host cell to
be active metabolically (in S phase of cell
cycle)?
• 2. How does SV40 insure that its host cell
is active metabolically?
• 3. Is SV40 mRNA transcription and DNA
replication similar to its host cell?