Replication of Large DNA Virus Herpesvirus,Poxvirus

Family Herpesviridae “creeping” spread of rash & vesicle lesions Widely found in nature – plants, fungi, animals, humans Highly infectious Infections – acute, persistent, transform Eight Human herpesvirus (HHV 1-8) Also primate, bovine, equine, swine, murine, avian herpesvirus

Herpes Simplex Virus (HSV-1) Infect mucous membranes and skin HSV-1: mainly oral & facial area Latent in neurons

Herpes Simplex Virus -2 (HSV-2) Mainly genital area Most infections are asymptomatic Symptoms of genital lesions soon after exposure, last ~10 days Latent in neurons, most have recurrent episodes within first year Mother with active infection may transmit to newborn during delivery

Genital Herpes: USA Epidemic Estimate >20 million infections Sexually Transmitted Diseases (STD’s) –Increasing since 1960’s –Social change (sexual freedom, changing moral standards, sex outside of marriage) –Birth control pill developed (non-barrier contraception) –Difficult problem for Public Health

Varicella/Zoster Virus (VZV) One virus, two diseases Varicella – chickenpox Latent in neurons Zoster – shingles, uncommon reactivation along nerve trunk in adults

Epstein-Barr Virus (EBV) Infectious mononucleosis Infects B lymphocyte, epithelial, fibroblast cells Latent in lymphoid tissue Co-carcinogen – Burkitt’s lymphoma, nasopharyngeal carcinoma

Human Cytomegalovirus (HCMV) “giant cells” in culture syncytia forms multinucleated cell Infects monocyte, lymphocyte, epithelial cell Latent in lymphoreticular cells USA – leading viral infection of fetus/newborns

Human Herpesvirus 6 (HHV-6) Exanthema subitum (roseola) Common rash in young children Infects lymphocytes

HHV-7 Isolated from lymphocytes of AIDS patient “orphan” virus No associated disease

HHV-8 Infects lymphocyte, vascular endothelial cells Viral DNA found in Kaposi’s sarcoma tissue of AIDS patients Co-carcinogen for Kaposi’s sarcoma

HSV-1 Envelope with surface projections, 200 nm Tegument (matrix) structure between capsid and envelope Icosahedral capsid, 130 nm Core with virus DNA wound in cylinder

HSV Genome: dsDNA Linear, one strand has nicks, 150 kbp Two unique components (U L, U S ) Terminal and internal repeat sequences Highly conserved “a” sequence at both ends (used for genome recognition and insertion into capsid)

HSV Genetic & Transcription Map

HSV: Attachment/ Entry Viral surface glycoproteins Host cell heparan sulphate proteoglycans Viral attachment blocked by polycations (polylysine, neomycin) Fusion of viral envelope with cell plasma membrane Capsid into cytoplasm Release of VHS (virion host shut-off) tegument protein that degrades cell mRNA in cytoplasm

HSV: Uncoating Viral capsid transported to nuclear membrane Release of DNA into nucleus Viral tegument protein αTIF (trans- inducing factor) transported into nucleus activates virus transcription

HSV: mRNA Transcription DNA genome circularizes Promoter/enhancer sites activated by viral αTIF and cell DNA-binding proteins (Oct- 1, SP1) Transcription from both DNA strands, bidirectional (clockwise, counterclockwise) Uses cell RNA pol II

HSV: Regulated Gene Expression Immediate-Early – α gene products, mainly regulatory Early – β gene products, mainly viral enzymes and proteins for DNA synthesis Late – γ gene products, some regulatory, mainly structual proteins

Cascade of HSV Transcription

DNA Replication (Rolling Circle) Synthesis of DNA in a long strand (head-to- tail concatemers) Viral enzymes Nick DNA strand, ssDNA rolls off Continous and discontinous (Okazaki fragments) DNA replication Concatemers later cleaved into genome size (recognition of “a” terminii)

HSV: Assembly Viral proteins transported into nucleus, assemble into capsid Viral DNA “head-full” insertion into capsid

HSV: Release Viral “primary” tegument protein associate with viral glycoprotein, buds through inner & outer nuclear membrane, releasing capsid into cytoplasm Capsid migrates to tegument proteins and picks up envelope by budding into exocytic vesicle Virus inside vesicles of cytoplasm; either remain cell associated or “secreted” to outside

Latent Infection Virus ascend up sensory nerve to neuron Viral DNA with cell histones and established in host cell as “episome” Expresses LAT (latency- associated transcripts) No infectious virus replication May be reactivated (immune suppression, stress, injury, UV light, hormone)

HSV Infection: Productive vs Latent

Reading & Questions Chapter 17: Replication of Some Nuclear-Replicating DNA Viruses

Class Discussion – Lecture 11a 1. How does HSV upon release of its DNA genome insure that it will be transcribed? 2. Like a good friend, HSV and its host cell have a lifetime relationship. How is this possible?

Family Poxviridae Viruses of vertebrates and insects Large “brick” shape, 200x300 nm –External, inner envelope –Lateral bodies –Complex coat of tubular structures Replication occurs in cytoplasm Benign tumors in experimental hosts

Human Poxviruses Characteristic rash and “pocks” Variola – smallpox –Transmitted by inhalation and infects respiratory tract, systemic infection –eradicated by WHO vaccination (1977) Vaccinia – “cowpox” lab recombinant used for vaccine Molluscum contagiosum – localized lesions, transmitted by contact

Occassional Poxvirus Zoonosis to Humans Localized lesions Transmitted by contact Orf – sheep, goat Cowpox – rodents, cats, cows Monkeypox – monkeys, squirrels

Vaccinia Virus Genome: dsDNA Linear, 186 kbp Covalently closed ends (“hairpin” loops) Inverted terminal repeats (10 kbp) Conserved central region Genes code for enzymes needed for RNA/DNA synthesis

Vaccinia Virus: Entry/Uncoating Fusion of virus with plasma membrane or entry by endocytosis Release of viral core into cytoplasm Viral proteins shut off host functions Further uncoating leads to “early” viral transcription/proteins in cytoplasm

Vaccinia Virus: Expression of “Early” Genes Virus core brings in enzymes required for viral transcription Half of genome is expressed from “early” gene promoters (activated by viral DNA binding proteins) Express enzymes needed for DNA replication

DNA Replication Occurs in cytoplasm Nick at end creates a free 3’ OH, self-priming DNA synthesis displaces parent strand, two genome concatemer (tail-to-tail) Continued DNA synthesis displaces two genome strand concatemer (tail-to-tail, head-to-head) Cleaved into two genome lengths Fill in and ligate ends into dsDNA, closed ends

Vaccinia Virus: Expression of “Late” Genes Switch due to viral regulatory proteins and configuration of newly replicated viral DNA Use of “late” promoters Expression of some enzymes, mainly structual proteins

Vaccinia Virus: Assembly and Release Sequential developmental stages in cytoplasm Viral membrane form crescent and circular structures Nucleoprotein mass forms with immature envelope and buds through golgi membrane for envelope Release by budding through plasma membrane

Vaccinia Virus Replication Cycle

Recombinant Vaccine Poxviruses have high recombination rate Dual infection of vaccinia virus + recombinant plasmid cloning vector with foreign virus gene Use of recombinant vaccinia virus + foreign gene for possible protective vaccine

Smallpox Virus: Potential Terrororist Weapon? Susceptible population Easily transmitted by inhalation Highly virulent strains (up to 40% mortality) Smallpox virus stored in two Public Health Labs (USA, former Soviet Union) Fear? Best defense?

Reading Chapter 18: Replication of Cytoplasmic DNA Viruses

Class Discussion – Lecture 11b 1. What would you postulate for the origin of Poxviruses? 2. If a terrorist ask your suggestion for a biological agent, would you tell him to go buy a herpesvirus or a smallpox virus from an underpaid government research microbiologist ? 3. Since smallpox has now been eradicated, would it be a good or bad ideal to destroy the remaining virus samples in the U.S.?

MICR 401 Final Exam Tuesday, Dec. 4, :30 – 3:00pm Papovavirus thru Hepadnavirus Case Study and Questions #9-15 Lecture & Class Discussion Questions, Reading & Chapter Questions Exam: –Objective Questions (MC, T/F, ID) –Short Essay Questions