5 - DNA replication/retrovirus (MICROM 450)

General basics of DNA replication

1. Viral protein binds to DNA origin of replication

2. Viral complex unwinds (helicase)

3. Nucleic acid primer laid down to initiate DNA synthesis

4. Polymerase complex templates bases onto new DNA strand (small DNA viruses use host DNA polymerase but larger ones encode their own)

Small DNA DdDp

Small viruses depend on host DdDp but a viral protein is still required to direct initiation of replication

**require dividing cells to replicate since they can increase expression of necessary replication complexes

Large DNA virus replication

Encode their own DdDp & can replicate in sub nuclear compartments to create virus factories

**allows replication in non dividing cells w/o relying on host cell division but dividing cells still preferred

Origins of replication

1. origin or rep is where DNA replication is initiated

   1. many origins bc chromosomes r so long

   2. can be internal or end of genome

2. Proteins involved:

   1. origin binding protein initiates replication by binding to sequences from origin of rep

   2. internal binding proteins have helicase activity to unwind so polymerase can access

-small DNA viruses don’t have polymerases but do encode their own proteins!

-diff types of viral orgins/site where synthesis begins

What kind of primer does DdDp need to start replication?

Eukaryotic cells use small RNA primers to start rep

• some use protein w OH group

• ssDNA viruses use ssDNA that is folded back on repeat regions to initiate, therefore primer is independent

What are the 2 main mechanism for DNA replication

1. Replication fork: initiated from RNA primer, origin of rep required, DNA viruses have multiple origins

   1. Ex. papillomaviruses, herpes, retroviral proviruses

2. Strand displacement: does not use RNA primer, terminal binding protein initiates

   1. adenovirus, parvoviruses, poxviruses

Small circular DNA virus replication

1. viral origin protein binds to origin on viral DNA to open up DNA via helicase activity

2. RNA primer is put down & host polymerase bind & extends DNA from primer

3. leading strand reads around the circle

4. laggin strand lays down regular primer & initiates okazaki fragments

Theta replication

Replication fork continues bi-directionally around entire genome until 2 circles are completed & seperate to create 2 new genomes

**NUMBER OF GENOMES EXPAND RAPIDLY

Herpesviridae: Rolling Circle Replication

Viral genome packaged in capsid as linear DNA to be extruded into nucleus via nuclear pore. Host ligases connect two ends of viral DNA to create closed circle then transcribed by host RNA polymerase & later replicated

**DNA GENOMES MUST BE CLEAVED TO BE PACKAGED INTO VIRIONS

DNA replication; strand displacement

Adenovirus-

• covalent linkage of dCMP-OH of serine in terminal protein by viral DNA polymerase

• continuous replication of bottom strand; after first 3 bases, 3 base shift backwards to generate exact ends (GTAGTA)

• top/displaced strand is coated w ss binding protein & circularizes through annealing of repeats at ends

  • allows TP to bring in polymerase & begin next strand

• top strand then replicated similarly to bottom strand (starting at ds region)

How can End Replication problem be overcome?

Eukaryotic chromosomes have telomeres added to ends to eliminate loss of DNA ends during replication & protect genome

**strand displacement limits this issue with 3 identical base shift

• circular genomes have no ends so they don’t have this issue

Why do DNA viruses replicate in the nucleus?

• Necessary cellular components, transcriptional machinery

• viral replication proteins must have nuclear localization signals

• DNA in cytoplasm signals induction of innate immunity

EXCEPTION: poxviridae encode DNA dependent RNA polymerase and its in the nucleus

Sub cellular replication

DNA viruses replicate in factories in the nucleus

Retroviruses

• retroviral genomes are flanked by identical long terminal repeats (LTRs)

• 3 main clusters: GAG core protein capsid, Pol polymerase, Env envelope glycoprotein & associated entry proteins

• complex retroviruses have additional accessory genes w other replication functions

Long Terminal Repeats (LTRs)

Important for induction of RNA synthesis from genome (like a promoter for transcription)

• repeats are important during reverse transcriptase step to generate full length DNA genomes

• repeats are longer than genomic RNA repeats

**retroviruses integrated into host genome to use host cell machinery (DdRp) to make RNA genomes BUT use LTR as promoter to initiate transcription

Retrovirus polymerase

Contains 3 activites:

1. RNA dependent DNA polymerase

2. RNAseH activity to seperate DNA from RNA strands

3. make 2nd strand of DNA, so they have DdDp activity too

What 2 additional enzymes does retrovirus polymerase encode

1. integrase: integrates dsDNA into host genome

2. protease: protein processing

Retroviruses replication cycle

1. capsid enters cytoplasm & is permeabilized (RNA stays inside capsid to bind to receptor)

2. RNA is reverse transcribed then made into dsDNA which travels to nucleus

3. Viral DNA is integrated into host genome

4. nearly full length transcription form LTR promoter occurs making Gag-Pol & RNA genomes

5. Spliced RNA made (makes envelope proteins)

6. RNA, capsid proteins & envelope proteins get together at cytoplasmic membrane

Retrovirus transcription

• host transcription machinery used to make retrovirus transcripts & then proteins

  • LTR promoter, RNA strand comes out shorter than DNA strand

Reverse transcriptase

• RT primed by tRNAs (in capsid in cytoplasm)

• RT initiates DNA synthesis toward 3’ end & switches to other 5’ end after 100 bases (allows for creation of both terminal repeats)

• + DNA strand then templated (using DdDp)

• DNA then transported to nucleus where integrase incorporates into host DNA

Hepadnaviridae replication: DNA retrovirus

Only human Hep B virus that infects liver/liver disease or cancer

Virus packaged as RNA but inside the virion is reverse transcribed to DNA before its released

• viral dna introduced into cells & makes circular dsDNA. dna transcribed to make viral genome template for reverse transcription. DNA doesn’t integrate but viral polymerase makes RNA to package & reverse transcribe in capsid

What does Hep B virus package in its capsid?

RNA, polymerase with RT, & deoxyNucleotides to make DNA

Why is Hep B virus dangerous?

Virus doesn’t lyse the cell, it buds out through golgi vesicle transport bc its not cytotoxic

**keeps producing more virus without killing cells

KEY CONCEPTS

-Small DNA viruses use host DNA polymerase, larger encode their own while being able to replicate in non dividing cells

-origin of rep binding proteins bring the polymerase to genome to initiate rep

-diff types of primers for DdDp used by viruses

-some viruses use replication forks to rep their own DNA, others use strand displacement

-viruses must be able to rep their entire genome + its ends

-retrovirus use host transcription machinery to make their mRNAs & their genome

-retroviral genome is shorter than integrated DNA genome