9 - DNA Virus Replication Flashcards
dsDNA viruses
- Herpesvirus
- Papillomavirus
- Polyomavirus
DNA viruses
Can cause productive lytic infections, cellular transformation, or latent infections
Lytic infection
The host cell dies as a result of producing progeny virions
Cellular transformation
- Host cells begins to be transformed into a cancer cell
- Cell becomes less responsive to physiological cues that regulate cell growth, differentiation, and death
- Can be an evolutionary dead end for the virus, if no progeny virions are produced
Latent infection
Viral genome is mostly quiescent and no virions are produced for a period of
time that is much longer than the duration of a lytic cycle (ie. up to the lifetime of the host)
Virus replication sites
- Cytoplasm: Most RNA viruses (besides retroviruses & orthomyxoviruses)
- Nucleus: Most DNA viruses plus orthomyxoviruses (besides Poxviruses)
Viruses that replicate in both cytoplasm and nucleus
- Retroviruses
- Hepadnaviruses
mRNP
Mature mRNA in the nucleus is exported to the cytoplasm through the nuclear pores as a messenger ribonucleoprotein (mRNP) complex
What do baltimore class 1 (dsDNA) animal viruses rely on for geen expression
Host transcription machinery
Class 1 reliance on host transcription machinery
- Most human cells do not divide
- Viruses do not replicate well in quiescent cells
- Viruses must induce host cell replication proteins (directly associated with virus size)
- Virus-encoded early gene products regulate this, and put cells into synthesis (S) phase
Protein required for synthesis
- DNA polymerase and accessory proteins
- Origin binding protein, helicases
- Exonucleases
- Enzymes of nucleic acid metabolism (eg. Thymidine kinase)
Viral origins or replication (Ori)
- AT-rich DNA sequence segments recognized by viral origin recognition proteins
- Viral genomes may have up to 3 ori
dsDNA bi directional replication
- Most nuclear dsDNA viruses replicate via this mechanism (in eukaryotic host cell nucleus)
- DNA replication begins at ori
- Topoisomerase unwinds dsDNA at ori
- ssDNA-binding proteins cover the ssDNA created in the replication bundle
- RNA primase synthesizes short RNA primers that are then used by the DNA polymerase to prime DNA synthesis
Polyomaviruses
- Small DNA viruses that drive cells into S phase
- Associated with post organ transplant kidney malfunction and merkel cell carcinoma
- Smallest dsDNA viruses that infect animal cells
- Non-enveloped spherical virions
- 4- to 5-kbp circular dsDNA genome
Prototype polyomavirus
Simian vacuolating virus 40 (SV40)
SV40
- Forces the host cell to express S phase genes and uses large T antigen and host proteins for genome replication
- Host cells susceptible to infection by SV40 are typically
in G0 prior to infection, which means that they do not
contain nuclear DNA replication proteins
-The virus must force the cell to express S phase genes in order to replicate its genome - Changes in gene expression are accomplished by the early nonstructural proteins large T antigen and small t antigen
SV40 large T and small t antigens
Multiple functions including manipulation of cell cycle and of apoptotic pathways
Rb (retinoblastoma) protein
- Controls entry into S phase
- Rb loss is associated with tumours (Rb is a tumour suppressor gene)
Replication of SV40 genomes
- Six large T antigen proteins form a hexamer, two hexamers bind ori in a head-to-head arrangement
- The hexamers have helicase activity that facilitates melting of the DNA at the ori
- DNA replication then proceeds bidirectionally, with each large T antigen hexamer progressing in opposite directions around the template (also known as θ replication)
Crystal structure of a hexamer
Composed of large T antigen helicase domains and six Zn2+ atoms
Initiation of SV40 replication by large T antigen
- LT protein, T-Ag (the major early gene product of SV40) assembles at its binding
site, ori - Intrinsic 3’-5’ helicase activity of LT unwinds DNA bi-directionally
- Viral DNA synthesis occurs in 5’-3’ direction on both leading and lagging strands
- Copied genome is assembled in capsid
- Association of T-Ag with cellular p53 can be oncogenic
SV40 early transcription
Proceeds unidirectionally terminating approximately halfway around the circular
genome
SV40 alternative splicing
- Major mechanism by which SV40 encodes so many different proteins
- Results in three different mature early mRNAs that encode either small t antigen, large T antigen, or 17-kT protein
SV40 early transcript products
Accessory molecules:
- Large T
- Small t
- 17-kT
Late and very late transcript products
Structural proteins
p53
- Key protein regulator of apoptosis in response to DNA damage
- Both DNA damage and viral infections activate p53
- When p53 is activated, it becomes phosphorylated, which has two major effects
- DNA viruses block p53 in order to prevent apoptosis and enable phosphorylation of pRB
Two major effects of phosphorylated p53
- Stimulates cells to undergo apoptosis
- Prevents the pRB protein from becoming phosphorylated (if phosphorylated it would detach from E2F and transcription would proceed)
Why do dsRNA viruses prevent or delay cellular apoptosis
Provides the virus with sufficient time to complete its replication cycle while enabling phosphorylation of pRB forces the host cell past the G1 checkpoint
Large T antigen
- Large T antigen interacts with pRB, preventing pRB from interacting with E2F transcription factors and
thus de-repressing S phase genes. - The large T antigen also interacts with p53, which
prevents p53 from stimulating apoptosis.
Small t antigen
Interacts with the PP2A (phosphatase) complex and inhibits its activity, thereby preventing repression of S phase genes
Polyoma virus JC
- Can result in a fatal demyelinating disease of the CNS
- Progressive multifocal leukoencephalopathy (PML) in which oligodendrocytes are destroyed
- Can remain latent after infection and be reactivated
- B lymphocytes principal carries of JCV DNA
HPV
- Reproduction cycle causes warts and leading causes of cervical cancer
- Detected via pap smear
- Gardasil vaccine prevents against most common forms
- Spherical virions with circular dsDNA genome 6–8.5 kbp
HPV replication cycle
- Tied closely to differentiation status of host cell
- Infect stratified squamous epithelia (found in skin)
- Can only infect the living basal cell layer, meaning that an injury is necessary to allow the virion to bypass the upper layers of the epithelium
- The earliest genes are expressed in the lower layers of the epithelium, whereas the later genes are expressed in the more distal layer
HPV replication in stratified epithelium
- The cells in the two lowest layers of the stratified epithelium are actively reproducing, thus providing DNA replication proteins to the virus
- The remainder of the epithelium consists of differentiated cells that are not regularly dividing as they are pushed toward the distal parts of the stratified epithelium
- In order to accomplish vegetative genome replication the virus must force the quiescent host cells in the
midzone to express host DNA replication proteins (E6 and E7)
E6
Covalently modifies the p53 protein, targeting it for degradation, thereby preventing p53-triggered apoptosis.
E7
- Binds to pRB so that pRB cannot bind to E2F transcription factors
- Consequentially, the E2F transcription factors no longer repress the expression of DNA replication S phase proteins.
HPV 1, 2, 4 and 7
Common warts
HPV 1, 2, 4, 60 and 63
Plantar warts
HPV 6, 11
Associated with genital, oral and laryngeal warts
High risk HPV subtypes
HPV 16 and HPV 18
Poxviruses
- Largest dsDNA viruses that infect humans
- Causes smallpox
- Linear genome composed of 194 kbp of dsDNA with inverted terminal repeats that form covalently closed hairpin termini
- Replicate in cytoplasm not nucleus, therefore need virally encoded RNA polymerase, transcription factors, and DNA replication proteins, likely contributing to the large poxvirus genomes
Poxvirus structure
- Enveloped, brick-shaped or ovoid virion, 220-450 nm long and 140-260 nm wide.
- The surface membrane
displays surface tubules or surface filaments
Two distinct infectious virus particles of poxvirus
- the intracellular mature virus (IMV)
- The extracellular enveloped virus (EEV)
Vaccinia growth factor activity
- Vaccinia growth factor (VGF) mimics cellular epidermal growth factor
- Binds to cellular epidermal growth factor receptors and triggers cells to enter the cell cycle and proliferate
