Lec 15 Adenovirus Flashcards
Adenovirus
The 5 genera based on genome sequence.
Aviadenovirus : virus of birds
Mastadenovirus: Virus of mammals
> 51 SEROTYPES of humans BASED on FIBER PROTEIN
6 subgroups based on
HA, serotype, oncogenic potential, GC ratio
Adenovirus Virion Structure
ICOSAHEDRAL structure, NAKED, 70-100 nm
Capsid of 252 capsomers, 12 pentons + 240 hexons.
HEXONS common Ag (specify adenoviruses),
fiber protein type specific Ag
Linear dsDNA 13% of mass (10-20 X 106). Problems w/ replicating it since the DNA must circularize before replication using IRFs.
Inverted terminal repeats at ends allow ssDNA to form DS-panhandles by folding.
55 kD PROTEIN COVALENTLY ATTACHED to DNA required for REPLICATION
Viral Structural Proteins
11 viral proteins
2 basic building blocks:
- Hexons which are trimers of the protein II 240 hexons cover the 20 faces. Made from Protein 2
- Pentons base subunit. Each made of 5 penton proteins of protein 3, each which lies at the vertices of the icosahedron.
Protein 6 is part of vertices.
Adenovirus Attachment
Mediated by fiber protein, Ab to fiber protein is protective
As each SEROTYPE has unique fiber proteins,
the RECEPTORS DIFFER between serotypes and Ab protection is therefore serotype specific
Most subgroups bind CAR (Coxsackie and Adenovirus Receptor)
Some bind CD46 (part of Ig superfamily molecule normal function in the FORMATION of TIGHT JUNCTIONS)
In some groups penton base then binds to integrin family by RGD sequence and loss of fiber protein.
The cell entry pathway of adenovirus
The cell entry pathway of adenovirus.
- attachment of the fiber to the primary receptor, which is on most cell types and for most HAdV types is either CAR or CD46.
- clathrin-mediated endocytosis, which is facilitated by the penton base interaction with αv integrins.
- The virion begins to dissociate in the low pH environment of the early endosome
- releases the vertex proteins including pVI in the early or late endosome. Protein pVI has been implicated in disruption of the endosomal membrane, allowing the to escape from the endosome.
- The partially disassembled virion is transported by the microtubule motor dynein along microtubules to the nuclear pore complex.
- At the nuclear pore the viral DNA is imported into the nucleus.
Adenovirus Penetration
Difficult to separate from attachment
Penton base proteins associate with host integrins (avb5 and avb3) at the RGD site
Virus enters via receptor-mediated endocytosis
50% of virus are free in cytoplasm in 5 min. Time suggests virus escape endosomes prior to fusion with phagosomes, pH independent? (H+ protons are pumped into endosome)
Adenovirus Uncoating
Microtubules bind hexon protein move virus to nucleus, associate at Nuclear pore Complex (NPC).
DNA enters via nuclear pore after uncoating that starts with pentons exploding out
Viral DNA associates with nuclear matrix via terminal protein (tp), which initiates replication
Early events in infection
Early events in infection.
- Adenovirus at the plasma membrane (PM), peripentonal hexons are in red.
- Binding of pentons to integrins and receptors followed by phagocytosis. The endosome triggers cytokines to trigger interferons that will trigger antiviral proteins! Adenoviruses can negate this until the host responds more aggressively.
- A phagocytic vesicle (endosome/lysosome) and disruption of the adenovirus particle releasing peripentonal hexons, IIIa and VI and revealing the core.
- Rupture of endosome/lysosome and release into cytoplasm of hexon shell with a metastable core (virus DNA with TP, VII, V, Mu and possibly protease).
- Hexon shell at nuclear pore (NP) with core being released into the nucleus.
- Core in the nucleus targeting cellular chromatin.
Virus Replication Cycle
Divided into early and late events, which separate
early: taking control of host, late:DNA replication/virion synthesis into discrete events
Some early proteins are however made into the late stages and some late proteins are made at low levels prior to DNA synthesis.
Early Transcription and Protein Synthesis
3 roles
Early genes play 3 roles
- Take control of cell and move it to S phase of growth cycle to allow for DNA replication
- Allow for viral DNA synthesis by producing viral enzymes that are needed
- Inactivate host immune system. Negate apoptosis, interferons, attack loose viral particles, have outer IS cells kill foreign antigen displaying cells.
Usually the cell must be in S phase before this happens, also seen in Retroviruses.
Transcription map and gene products of adenovirus type 5
RNA splicing is used to generate several proteins. This virus helped us discover splicing!
Early genes transcriptional starts. If there is a gap before an arrow, splicing will happen for formation and polyadenylation.
Splicing works to give structural proteins to assemble visions (late genes)
Early genes in depth
Similar in all serotypes
4 sets of early genes E1 (E1A, E1B), E2 (E2A, E2B), E3, and E4
2 sets of delayed early genes IX and IVa2
2 sets of VA genes transcribed by host polymerase 3 (others by polymerase II)
All but VA produce multiple mRNA species due to splicing events
Both DNA strands equally code for early genes
Late genes on top or R-strand only. Both strands are used to produce different transcripts.
Early Gene Function
Viral E1A yields 2 immediate early mRNAs and 3 later mRNAs.
ElA binds retinal blastoma protein (E1A) and phosphorylates it to force E2f to enter S phase!
Trans-activators of viral genes and initiate host into S phase by working through common pRB systems to repress E2F complex.
Also activate CDK.
RB is the retinoblastoma family of cellular tumor suppressor proteins
E2F (regulates the adenovirus) E2 promoter, but also regulates many genes involved in DNA synthesis and cell cycle progression factors
In normal g1 resting cells CDK (cyclin dependant protein Kinase) phosphorylates RB and it can no longer bind
Normal cell: E2f binds Rb and HDAC (histone deacetylase), when E2f is released the cell enters the S phase
*E1A indirectly induces apoptosis by activation of p53
E1A also stabilizes and activates p53, which could induce apoptosis by up regulating p14Art, decreasing Mcm2 concentration so p53 accumulates.
E1B inactivates p53, tagging it w/ Mcm2 and degrading it in proteosome! Prevents apoptosis due to forcing change in cell cycle.
Other Early genes E2 E3 E4
E2 genes are important in viral DNA synthesis
E3 genes contribute to the virus hiding from the immune system by down regulation of MHC class I and prevents immune mediated induction of apoptosis
E4 most variable region of early genes with respect to function, trans-activate genome, increase viral mRNA transport from nucleus, assist in DNA replication
DNA Replication Pt 1
Requires host to be in S phase and accumulation of E2 gene products
Starts 5 hours after infection
Takes place in 2 steps
- Synthesis starts from either end in one direction replacing one original strand and forming a new duplex of one old and one new strand
- Remaining parental strand is then duplicated
DNA Replication Pt 2
A PRE-TERMINAL PROTEIN (tp) associates with viral polymerase and an E2 protein to form complex that binds to origin of replication (domain A).
Domains B and C of origin are not required but augment DNA replication.
The same complex replicates the DS-DNA and the SS-DNA strand displaced in the original step, following the same principals.
Pre-tp is later cleaved to tp.
Adenovirus DNA replication
Viral DNA is replicated by 3 viral proteins
- pre-terminal protein pTP (bluish oval)
One vDNA is inserted info nucleus, a pTP attaches to TP to prime DNA synthesis w/ DNA pol and during this, the top (parental) strand displaces ssBPs to form pearl necklace in 5 to 3’ direction.
The second parental strand is complexes w/ ssBPs and terminal repeats form a loop via base pairing of terminal repeats, the pTP protein synthesizes a complementary strand and the pTP is cleaved off to create TP at each 5’ end.
-DNA polymerase (orange circle)
-single stranded DNA binding protein
(green circles)
-Single-stranded DNA is circularized via the inverted terminal repeat
Late Gene Expression
Low levels of late genes transcribed prior to DNA synthesis.
-IVa2 gene products up regulate late genes
-Genes found on r-strand, long transcript made (>29,000 bp) by eliminating premature termination.
Late mRNAs produced by splicing
There are many spots where polyA tails are added, and many leader sites where splicing occurs 1 region and L1s to form different truncated versions. L3 can create 4 transcripts.
Virus Assembly and Release
Penton and hexon assembly starts in cytoplasm and assembled units move to nucleus
First empty capsid assembles
DNA enters via packaging molecule
If no DNA capsid falls apart
Virus release involves disruption of intermediate filaments (vimentin)
Large amounts of viral components collect in nucleus
Adenovirus kills by apopotosis aiding in Virus Release
E3 11.6 kDa “death protein”
Expressed at high levels late
Membrane glycoprotein predominately found in nuclear membrane
E4 orf4 can also induce death in a p53 independent manner
If expressed alone kill cancer cells but not normal cells
Adenovirus Distinct characteristics
Multiple mRNAs arise by extensive alterative splicing
Late transcripts made from a single strong promoter and five alternative splice sites
DNA synthesis primed by terminal protein uses viral DNA polymerase
Can cause tumors in experimental animals but not known to cause tumors in humans
Adenovirus are widely used as gene therapy and anticancer virus vectors
