Replication of Replication of Small DNA Small DNA

VirusVirus

Replication of Replication of Small DNA Small DNA

VirusVirus

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

• 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

Virus Infections

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

• 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: “Late” Proteins

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 proto-

oncogene 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?