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 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?