1. Adenoviruses

2. Characteristics of Adenoviruses Name originates from Greek word “adenas” which means gland, site from which were initially isolated Naked capsid with eicosahedral symmetry Fibers protruding from capsid facilitate binding on to host Genome is linear dsDNA, 30-36 Kbp 11 proteins are found in the virion Genome encodes for up to 50 proteins Over 50 human serotypes Most regions of genome are transcribed by cellular RNA PolyII

3. Characteristics of Adenoviruses They cause respiratory syndromes such as pneumonia Eye and gastrointestinal infections Not known to cause cancer in humans, only in experimental animal models Adenoviruses divided into groups –A-F –This classification is based on ability to agglutinate blood –We will be covering types 2, 5 (group C) and type 12 (group A)

4. Viral Attachment Attachment is mediated via “antenae”, the fibers protruding from capsid Knobs on antenae bind receptors on target cells The receptor in most cases is the CAR receptors (coxsackie and adenovirus receptor) Integrins behave as secondary receptors facilitating clathrin coated pit mediated endocytosis Virus enters host inside endosomes

5. Cytosolic Transport to Nuclear Pores Virus exits endosomes via a pH dependent mechanism Transportation occurs via microtubules Interaction occurs between hexon and microtubules Once at the pore, capsid disassembles and releases dsDNA genome into nucleus

6. Expression of Adenovirus Genome Approximately 50 genes are being expressed –Though complicated at first glance it has some logic to it 8 Transcriptional promoters and 12 Poly(A) signals 6 Early Transcription Units, E1A, E1B, E2A, E2B, E3 and E4 –E for early –That is why retroviruses infect dividing cells more efficiently 2 Intermediate IVa2 and IX 3 Late, L1-L5 The essence is 50 different mRNAs It is important to understand transcription concepts rather to memorize them

7. Expression of Adenovirus Genome For example one common promoter but different Poly(A) signals

8. Expression of Adenovirus Genome Alternative splicing yields a variety of translatable mRNAs Different promoters yield different mRNAs This is the reason why from only 13 transcriptional units you end up with 50 different viral proteins

9. Genome Encodes for RNAs That Never Get Translated VA genes never get translated VA stands for virus associated genes These genome segments are transcribed by cellular RNA Pol III –This enzyme is normally used for tRNAs and other small RNAs VA encoded RNAs inhibit PKR –PKR (ds RNA dependent protein kinase) PKR inhibition avoids destruction of virus PKR is involved in interferon signaling

10. The E1A Is Crucial for Viral Replication 2 mRNA exist that encode for E1A –First one yields a 289 a/a long E1A –Second one yields a 243 a/a long E1A These 2 proteins are powerful transcriptional activators for several viral and cellular genes Adenoviruses target non-dividing fully differentiated epithelial cells Therefore proteins that aid in DNA replication have to be induced E1A induces cells into S phase E1A is oncogenic, at least in experimental models

11. How Does E1A Work? It interacts with Rb and Rb family proteins –Rbs are tumor suppressive proteins Normally Rb is interacting with the E2F transcription factor E2F received its name from the fact that it was regulating the E2 promoter of adenovirus p107 and p130 have similar function as Rb E2F

12. E1A Indirectly Induces Apoptosis Through p53 p53 is a tumor suppressor transcription factor Normally Rb is interacting with the E2F transcription factor Activation of p53 is a sign of problems that leads to apoptosis Apoptosis is regulated death –Occurs when extensive DNA damage has occurred –Viral infection or other insult E1A increases expression of p14 Arf (mediated thru E2F transcription) ⇒ p53 is stabilized ⇒ apoptosis occurs E1A also ↓Mdm-2 ⇒ apoptosis Apoptosis is NOT good for viral replication E1B to the rescue

13. E1B Suppress E1A Induced Apoptosis Apoptosis is detrimental to viral propagation E1B produces multiple mRNAs 1 mRNA (2.2 Kb) produces 2 proteins (due to 2 different initiation sites) –19 KDa and 55 respectively The 19 KDa is a functional homologue of bcl-2 (an anti-apoptotic protein) Blc-2 homologue neutralizes bax (pro-apoptotic protein) ⇒ apoptosis ↓ 55 KDa protein binds directly to p53 minimizing apoptosis related to p53 transcription Ultimately cell survives longer, resulting in higher levels of viral progeny but ultimately dies from excessive virion production

14. E2 Genes Products Responsible for DNA Replication 3 essential proteins from E2 region are needed for DNA replication –ssDBP (single stranded DNA Binding Protein) from E2A region –Viral DNA polymerase from E2B region –pTP (pre terminal protein) from E2B region 1 st step in DNA replication is unusual –No mRNA primer for DNA polymerase –Instead pTP acts as the primer This replication repeats several times with NO net viral progeny!! Why? New ssDNA must be converted to dsDNA, otherwise no net gain

17. New ssDNA Completes DNA Replication 1 st ssDNA circularizes thru ITRs (inverted terminal repeats) The stem recreates the replication origin The pT/Polymerase bind completing replication ssDBP is displaced by polymerase during replication as it moves in the 5’ to 3’ direction 1 st step in DNA replication is unusual –No mRNA primer for DNA polymerase –Instead pTP acts as the primer

18. Alternative Pathway

19. Late Major Promoter Encodes 1 Long pre- mRNA Transcript Transcription from this promoter begins soon after DNA replication Produces one pre-mRNA from which all late mature mRNAs are produced Activity of this promoter ↑x100 due to –Newly synthesized genome has a more accessible “late major promoter” probably due to host trans factors binding –IVa2 polypeptide binds downstream, promoter cooperates with upstream enhancer, ultimately more efficient transcription 5 families of late mRNAs –18 mRNAs arise due to a variety of poly(A) sites and variable splicing sites “Tripartite leader” is common to all mRNAs All mRNAs have unique AUG translation sites –Unique reading frames ⇒ unique proteins

20. Advantages of Common pre-mRNA Coordinated expression of all late genes Common ‘tripartite leader’ allows for efficient transport to cytosol ‘Tripartite leader’ allows for ribosome shunting (no mRNA scanning) ⇒ translation occurs All cellular mRNAs cannot be translated due to dephosphorylated eIF4F by a viral protein

21. Therapeutic Applications of Adenoviruses Adenoviruses are already used in clinical applications/trials to deliver genes in diseased tissue They are appropriate due to ability to infect variety of cells Induce high expression of their genes Adenoviruses are used as vectors E1A and E1B are removed to avoid transformation of cells Adenoviruses are used for cancer/pathogen genes to invoke immune response ONYX-015 strain used to infect cancer cells and destroy them (mutant in E1B ⇒ no p53 inactivation) –Tumor cells do not have functional p53 –Viral replication leads to their destruction