replication of DNA viruses II

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this lesson about rest of DS viruses:

1. - normal DS DNA
2. - DS DNA that go thru retroviral stage

double stranded DNA viruses

1. double stranded
2. double stranded with RNA activity

3 families of DNA viruses are enveloped

- herpes, Pox, hepadna viruses

Pox viruses don’t need to replicate in nuclei

- they are independent bc have machinery they need to replicate

class I: double stranded DNA

subdivided into 2 groups:

• replication is exclusively nuclear
  • replication of these viruses is dependent on cellular factors
• replication occurs in cytoplasm
  • these viruses evolved / acquired all factors for transc./replication of genomes and are largely independent of the cellular replication machinery

double stranded DNA virus replication

diverse/more complicated replication mechanisms than RNA viruses

some require host pol, some encode their own replication factors

dsDNA viruses make use of several mechanisms to replicate their genome

1. bidirectional replication
2. rolling circle mechanism
3. strand displacement

DS DNA bidirectional replication

(GO OVER THIS)

bidirectional replication

• 2 replication forks established at replication origin site + move directions opposite of each other

DS DNA rolling circle mechanism

(GO OVER THIS)

produces linear strands while progressing in a loop around circular genome

DS DNA strand displacement

(GO OVER THIS)

method where one strand is made from template strand, complimentary strand is made from prior synth. strand

= dsDNA genome

During strand displacement replication, only one strand is replicated at a time. This synthesis releases a single-stranded DNA, which in turn is copied into a double-stranded DNA.

DS DNA viruses transcription

3 step process to make mRNA

1. transcription preinitiation complex
   • binds to DNA upstream of site where transc. begins -> allows recruitment of host RNA polymerase (RNA pol can be from self or from host, depending on if the virus has its own things needed to replicate)
2. use negative srand (non coding strand) as template for synth. mRNA strands
3. RNA pol terminates transcription upon reaching stop signal

ds DNA cviruses

Ds DNA virus does 2 things with its genes

- make mRNA -> translated in ribosomes of infected cell (NO viruses have their own ribosomes for translation)

- replicates its ds genome – make copies

what ribosomes must ALWAYS be used for translation?

HOST CELL RIBOSOMES

viruses NEVER have their own ribosomes

DS DNA viruses subdivided into 5 families

1. polyomaviridae
2. papillomaviridae
3. adenoviridae
4. herpesviridae
5. poxviridae

infect mammals and us

polyomaviruses

• small
• nonenveloped
• circular DS DNA
• widespread in nature
• latent in healthy hosts after primary infection
  • illness occur in immunocompromised ppl
  • Immunocompetent healthy host-> viruses may internalize + be latent - we are exposed to them - whether we get sick or not depends on our immune strength
• ex: human polyomaviruses BKV and JCV cause hemorrhagic cystitis and progressive multifocalleukoencephalopathy
  • In ppl recipients of organs, they are subjected to immunosuppressed states, and these viruses can make them sick bc of lowered immune system
• ex: merkel cell polyomavirus may cause merkel cell carcinoma
• ex: SV40 can cause tumors, usually is latent though if healthy

papillomaviruses

• small
• nonenveloped
• icosahedral circular DS DNA
• infect humans, cattle, dogs, rabbits, monkeys, and some more.
• ex: HPV (human papillomaviruses) infections are usually subclinical or can cause benign lesions
  • but infections by high risk types can lead to cancer
• genome:
  • early region
    • code for transforming proteins that mediate oncogenic properties of the high risk HPVs (E6/E7 genes)
  • late region
    • encodes for major L1 capsid protein -> accounts for most of virion mass + mediates viral attachment (what vaccines are based on)
  • noncoding long control region
    • has regulatory elements for viral DNA repl./transc.

mechanism by which cellular transformation of E6/E7 genes of high risk HPV work?

E6 protein complexes with tumor suppressor protein P53 and targets it for destruction

E7 complex w/ tumor suppressor protein (Rb) -> regulates expression of genes that activate cell cycle

(normally p53 and Rb inhibit cellular proliferation)

• E6 protein made by HPV inhibits function of p53
• E7 protein made by HPV inhibits function of Rb

in HPV infected cels, HPV oncoproteins lead to

continued cell proliferation without time for repair of DNA damage

leads to genetic instability + accumulation of addl. cellular mutations + chromosomal changes

-> thus infections with higher risk HPVs prepare ground for cellular genetic alterations that underlie cervical cancer

HPV proteins lead to cont. cell proliferation

adenoviruses

• medium sized
• nonenveloped (naked)
• icosahedral nucleocapsid
• isolated from human adenoids
• range of illnesses caused
  • ex: mild resp. virus (common cold), conjunctivitis, deadly disease in ppl w/ weakened immune system
• genome
  • linear, nonsegmented DS DNA (30-40 genes)
  • heavily relies on host cell for survival/replication
  • relies on cellular apparatus for transcription, but possesses various mechanisms that regulate viral gene expression
• ex: trans-acting transc. activators like E1A protein, post transc. regulation of expression, etc.
• infection of cells divided into 2 stages (early/late)

gene therapy - adenoviruses

• adenoviruses - popular viral vector for gene therapy due to ability to affect replicating and non replicating cells
• code for proteins w/o integrating into host cell genome
• use as vehicle to administer targeted therapy in form of recombinant DNA/protein
• found to work in monogenic diseases + cancer

Used as vector

Adenoviruses big enough to accommodate transgenes

Use to repair or internalize 1 particular gene that may be missing, mutated, etc.

Used as vehicle to administer targeted therapy

Oncolytic adenoviruses -> lyse the cells as cancer treatment

vaccines - adenoviruses

• adenoviruses used to make viral vector COVID-19 vaccines
• Ad5 - vector to transport surface protein gene of SARS-CoV-2
• goal is to genetically express the spike glycoprotein of covid

Vaccines

- some COVID 19 vaccines use adenoviruses as vectors to introduce spike

Adenoviruses good for gene therapy + vaccines

herpesviruses

• enveloped
• large
• DS linear DNA
• 3 groups
  • alpha herpesviruses
    • type 1
    • type 2
    • chickenpox virus
    • short cycle, induce cytopathology, broad host range
  • beta herpesviruses
    • cytomegalovirus and human herpesvirus 6/7
    • long cycle, restricted host range
  • y herpesviruses
    • epstein barr virus and human herpesvirus 8 (kaposi sarcoma virus)
    • very restricted host range
  • all herpes virus can establish lifelong latent infection w/ specific tissues (characterized for each virus)

herpesvirus transc., gene replication, and capsid assembly occur in

host cell nucleus

genes replicated in specific order

1. immediate-early genes
2. early genes
3. late genes

immediate-early genes encode

regulatory proteins

early genes encode

enzymes for replicating viral DNA

late genes encode

structural proteins

how herpesvirus get out of cells and what happens in latent cells

• virion progeny transported to cell membrane via golgi complex + host cells die as mature virions released
• in certain cell types, virus stays in lifelong latent state
  • ex: latency of chicken pox (in nerves) -> CAN get shingles when older
  • neurons, monocytes, lymphocytes
  • latent viral genome may reactivate at any time

transmission of HSV 1 and 2

1 - oral

2 - genital

control of herpesvirus infection

• prevention
  • vaccine for chickenpox
  • HSV 2 and cytomegalovirus only have passive immunity
  • avoid contact when lesions are visible
• treatment
  • acyclovir for hsv 1, 2, and chickenpox
  • etc, see slide

herpesviruses (80 genes)

• encode many enzymes of DNA metabolism + trans-acting factors that regulate temporal expression of virus gene, controlling phases of infection
• transcription of the large, complex genome is sequentially regulated in a cascade fashion

Not as independent as poxvirus

Encode enzymes involving DNA metabolism

Expression is regulated

Cytomegalovirus – related to microcephaly, Etc.

herpesvirus gene expression

poxvirus

• isolated from birds, insects, reptiles, marsupials, mammals
• complex enveloped virions
• large - visible by light microscopy
• ds DNA genomes
• terminal hairpins linking the 2 DNA strands -> single polynucleotide chain
• ex: variola virus (VARV) - cause smallpox (extinguished w/ vaccines). molluscum contagiosum and monkeypox most frequent in world
• 10 infect ppl
• zoonoses
• minor abrasions of skin
• encode all enzymes needed for RNA and post. transc. modification of mRNAs, and DNA synth.
• replicate in cytoplasm using only host ribosomes
• many enzymes packaged (all it needs) in virus particle -> so replication occur in cytoplasm under viral control

Poxviruses

- best known – variola virus – cause smallpox

- extinguished w/ vaccines

Large – large enough to see on light microscope

DS DNA genome w/ terminal hairpins

10 poxviruses infect humans

Poxviruses – zoonotic (from animals)

Poxvirus outbreaks recently due to cessation of vaccines

- bc they are very similar to each other

unlike other DNA viruses that rely on nucleus, poxviruses replicate entirely

in the cytoplasm of infected cells

poxviruses evade ___ well

innate / adaptive immune responses

using proteins encoded by their large genome

poxvirus gene expression

by virus enzymes associated w/ core of particle + divided into 2 phases

early genes

• 50% of poxvirus genome
• expressed before genome replication

late genes

• expressed after genome replication in the cytoplasm
• but expression is dependent on virus-encoded rather than cellular transcription proteins

poxvirus replication cycle

additional effects of DNA viruses: on the cell cycle

• p53 inhibitors
  • SV40 large T (polyoma)
  • adenoma E1A
  • HPV E6
• RB inhibitors
  • SV40 large T (polyoma)
  • Adeno E1B
  • HPV E7

Addl effection of DNA viruses

- tumor suppressors – breaks in cell cycle

- viral proteins interact w/ inhibitors

viral effects on host cells

viruses can have one of several diff effects on their cellular hosts

• abortive infections
• cytolytic infections
• persistent infections

abortive infections

virus cannot go in cell (depends on receptor)

if infect cell unable to receive virus -> abortive infection, does nothing to the cell

may result when virus mistakenly infects a cell that does not permit viral replication (tropism)

cytolytic infections

lead to cell lysis and release of large numbers of virus

persistent infections

may be productive, latent, or transforming

• exit thru budding
• can be latent
• can be transforming - oncogenic, transforms the cell

coordinated replication of viral components

1. expression of viral regulatory proteins (immediate early genes)
2. expression of viral genes involved in replication (early lytic genes)
3. replication of viral genome
4. expression of late lytic genes (capsid, envelope)
5. assembly and egress

DS DNA with RNA intermediate

hepadnaviridae

• hepadnaviruses - group of DNA viruses infect hepatocytes + can cause liver injury and hepatocellular carcinoma (HCC) in mammals/birds
• Hep B (HBV) - cause acute/chronic Hep B, cirrhosis, and HCC in humans
• small
• enveloped
• partially DS DNA (3-3.4 Kb) replicated by reverse transcription of RNA intermediates
• use overlapping reading frames (ORF)
  • to encode
    • envelope
    • nucleocapsid proteins
    • polymerase w/ domains that having priming, reversetranscriptase, and RNAse H

what family of viruses is DS DNA with RNA intermediate?

hepadna viridae

genome:

small genomes of partially DS, partially SS circular DNA

genome = 2 strands

• 1 longer (-) sense (purple)
• 1 shorter (+) strand of variable length (orange)

2 ends of long meet but not bonded together, and the shorter strand overlaps over the divide

replication

• viral DS DNA convert to covalently closed circular DNA by viral polymerase (in nucleus)
• replication involve RNA intermediate
• 4 main reading frames encoded = virus has 4 genes that encode 7 proteins
  • core capsid protein C
  • viral polymerase P
  • surface antigens (preS1, preS2, S, X, HBeAg)