# Test 2, Lecture 13 Description of Adenovirus virion Naked icosahedron capsid with spike-like fiber proteins connected to penton proteins at each vertex. The fibers are non-covalently bound but high affinity. The icosahedron's faces are made up of hexon proteins. Size of Adenovirus genome 36 kb Adenovirus binds to these cell receptors HCAR (primary), MHC I (some strains this is the primary) Adenovirus binds to these coreceptors on the cell Integrins, which are bound by the penton proteins. General steps of initial Adenovirus infection Fiber attaches to receptor (HCAR and/or MHC I). Pentons bind integrins. Virion enters via receptor-mediated endocytosis, released into cytoplasm. Bursts the endosome before lysosome can reach it. Uncoats upon reaching the nuclear membrane. When released into the cytoplasm, Adenovirus' hexon and penton proteins are... cytotoxic. Hexon causes rounding up and detachment, penton inhibits translation and transcription, then replication, shift to viral synthesis. RNA Pol II transcribes this Adenovirus immediate-early gene E1A Adenovirus E1A yields 3 different proteins because of this alternative splicing Purposes of Adenovirus E1A regulatory proteins Enhancer for transcription of delayed-early genes; represses host MHC I transcription (no CTL killing); induction of viral DNA replication; interaction with cellular oncogenes to transform cells and stimulate the cell cycle Adenovirus E1A leads to expression of these E1B, E2A, E2B, E4, E5 Adenovirus E1B does this Interacts with oncogene products to transform cells, with E1A stimulates delayed-early expression Adenovirus E2A and E2B do this DNA replication. DNA binding phosphoprotein (E2A); Single-stranded binding protein (E2B), DNA polymerase (E2B), Pre-terminal protein (E2B). Adenovirus E3 does this Prevents transport of MHC I through Golgi (no CTL killing), so MHC is not on cell surface How Adenovirus solves the problem of replicating its genome It is linear dsDNA, but must solve problem of priming at the ends. It uses a 80-kD pre-terminal protein with a cytosine that pairs with the 3' G of one strand of the dsDNA. That serves as a primer which allows for continuous replication of one entire strand, while displacing the other strand. After replication of the strand, the pre-terminal protein loses a 25-kD fragment to become a 55-kD fragment that remains attached to the strand. The displaced strand is then replicated because it also has a 3' G that is able to pair with the 80-kD pre-terminal protein with the cytosine. Adenovirus has this type of DNA replication Asymmetric semi-conservative DNA replication (one strand at a time). Adenovirus late gene expression occurs after... DNA replication Adenovirus's late gene expression shows that a single late promoter is involved which produces a single transcript 30,000 bases long. This transcript leads to translation of 15 different proteins, which led to the discovery of... splicing and alternate splicing Heteroduplex mapping of genes Common technique to map genes; hybridize mRNA to chromosome, look for where the mRNA is bound with EM; locates the position of gene How splicing was discovered with Adenovirus mRNAs The heteroduplex map showed that the mRNAs were hybridized to the chromosome, but large loops had formed indicating sections of transcribed DNA that were spliced out in the final transcript. The fact that multiple looping patterns were found showed that the transcripts could be spliced in different ways (alternate splicing). poly A choice Multiple sites on the transcript where the poly(A) tail can be attached can create different mRNAs. leaders leader exons are shared in all finished mRNAs The 5' ends (leaders) are shared in all finished mRNAs because... they contain the ribosomal recognition sites (i.e. Kozak sequence) inclusion bodies masses of viral proteins that form in nucleus; good for diagnosis Adenovirus' assembly and escape Assembly is in nucleus; assembly is very inefficient (leaves inclusion bodies); release is slow, mostly after cell death; leads to inapparent persistent infections in conjunction with immune suppression Adenovirus VA RNAs are transcribed by this RNA Pol III, because they are small RNAs Adenovirus VAI and VAII are... translational enhancers; because without them, viral translation does not occur VA RNAs Virally-associated RNAs re expressed by Adenovirus at low levels early, at very high levels late. They are small RNAs (160 nt) that are not translated, but are required for translation to occur. Loss of VA RNA causes inhibition of translation initiation (eIF-2 activity was interrupted; interruption caused by interferon). Purpose of large number of VA RNAs is to overwhelm DAI with excess dsRNA as countermeasures for interferon. Purpose of VA RNAs... Large numbers of VA RNAs overwhelm DAI with excess dsRNA as countermeasures for interferon. Interferon Substance produced by infected cell that induces "anti-viral state" in neighboring cells; infected cell secretes it; producing cell is not protected but neighboring cells are; foreign genome induces expression and secretion; Types of interferons Type I (IFN-alpha and IFN-beta) are made by all types of cells; Type II (IFN-gamma) is made by lymphocytes and secreted by T_H cells as lymphokine. Interferon activity Bind to cell surface receptors; JAK-STAT signal transduction cascade induces 2 pro-enzymes; Double-strand activated inhibitor (DAI), when activated by dsRNA, blocks translation; Oligo A Synthetase, when activated by dsRNA, degrades mRNA; together, they block protein synthesis so viruses can't multiply. DAI, when activated by dsRNA... becomes phosphorylated (to DAI-P). DAI-P can then phosphorylate eIF-2. When eIF-2 is phosphorylated, GEF cannot restore GTP to eIF-2-GDP to form eIF-2-GTP and initiate translation. RNA interference Introduction of dsRNA that corresponds to one side of an mRNA causes expression to be blocked by having the mRNA degraded. Cellular protection against genome invaders. Dicer ATP-dependent ribonuclease involved in RNA interference; it chops of dsRNA into fragments of 20-25 nt RISC RNA-induced silencing complex; responsible for destroying mRNAs when fragments from Dicer associate with it