Replication Replication of Large of Large

DNA VirusDNA Virus

Replication Replication of Large of Large

DNA VirusDNA VirusHerpesvirus, Herpesvirus,

PoxvirusPoxvirus

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 (UL, US)• 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)

HSVInfection:Productive

vsLatent

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, 2012• 1:30 – 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