1. Transcription of viral DNAs. Lecture 14 Flint et al. pp. 253 – 277.

2. General points For RNA viruses, most viral mRNAs are synthesized by Viral RDRP For DNA viruses, viral mRNAs are generally synthesized by cellular RNA polymerases. –Some exceptions: e.g. Poxviruses have their own DNA-dependent RNA polymerase. Transcription and expression of viral genes occurs in a strictly defined, reproducible sequence. Generally: –Genes for viral enzymes and regulatory proteins are transcribed early in infection. –Genes for structural proteins are transcribed later.

3. The relative simplicity of viral systems has enabled their use in elucidating the general properties of cellular RNA transcription What we know about transcriptional control is based on virology. Therefore, we must first digress back to cellular transcription.

4. Cellular transcription DNA dependent RNA polymerase Cells contain 3 DNA dependent RNA polymerases (see Table 8.1) RNA pol I: transcribes pre-rRNA; no known viral templates RNA pol II: transcribes pre-mRNA & snRNA: polymerase for most viral DNAs. RNA pol III: transcribes pre-tRNAs, 5S rRNA, U6 snRNA; polymerase for some viral DNAs.

5. Finding the right place to start The transcriptional machinery must: –Be directed to initiate transcription at the correct location on a DNA template (the transcriptional start site). –elongate through the entire gene –Be directed to terminate transcription at the correct location. All of these functions require the assistance of –Cis-acting sequences along the DNA –Trans-acting factors (accessory proteins)

6. Nuclear localization DNA dependent RNA polymerases located in nucleus Viruses that usurp these functions must localize their templates for this machinery into the nucleus. Three variations on this theme (Table 8.2) 1.Viral genome looks like a chromosome: -Associated with host-cell nucleosomes or viral proteins that look like nucleosomes. e.g. Papilloma- and Polyomaviruses 2.Viral ssDNA converted to dsDNA in viral particle. dsDNA then imported into nucleus. e.g. hepadnaviruses 3.Viral RNA reverse transcribed in viral particle. cDNA imported into nucleus and integrated into genome. e.g. Retroviruses.

7. Transcription by RNA Pol. II. At least 40 proteins required: Pol. II itself + accessory proteins. Accurate transcription initiated at the promoter. Promoter + additional DNA sequence that controls transcription = Transcriptional control region (TCR). –The adenovirus type 2 major late promoter was the first TCR ever recapitulated in vitro. Initiation is a multistep process: –Promoter recognition by RNA Pol. II –Formation of open initiation complex (unwinding) –Promoter clearance –3’ movement of complex away from promoter

8. Transcription by RNA Pol. II. Fig 8.2

9. Promoters (Fig. 8.1) Core promoters Contain all the information necessary for recognition of initiation start site. Direct RNA Pol. II complex to begin transcription. Contain TA-rich “TATA box” sequences. –Thermodynamically easy to unwind. –Located 20 – 35 bp upstream (5’) of start site. Contain Initiators Sort sequences Specify accurate but inefficient transcription

10. Promoters (Fig. 8.1)

11. Regulation of Pol. II transcription Transcription must be regulated: genes must be turned on and off in temporal patterns Viral gene expression: early and late genes Transcriptional regulation is controlled by: –Cis-acting sequences in DNA – both local and distal –Trans-acting factors – both protein and RNA Trans-acting factors specifically bind to cis- acting sequences to either –activators stimulate transcription –repressors prevent transcription

12. An enormous number of sequence- specific transcriptional regulators. Basic properties (Fig. 8.7) Modular organization – built from discrete structural and functional domains. DNA binding module – targets protein to a specific DNA sequence Activation (or repression) domain – interacts directly or indirectly with the RNA polymerase. Dimerization domain – activation tends to require interacts with other transacting factors. Provides a way to fine tune activity. –Homo-dimer: interaction of two copies of the same factor –Hetero-dimer: interaction of two different factor.

13. Modular organization of sequence-specific transcriptional regulators

14. Example: Regulation of HIV-1 transcription by host and virus encoded factors. The HIV-1 transcriptional control region: Located in the 5’ region of the proviral genome. Divided into 3 enhancer regions. –Promoter: Contains binding sites for TFIId and SP I factors: recruit RNA Pol II to promoter. –Core enhancer: binding sites for Nf-kB and Ets-1. These proteins are only active in growing T-cells, i.e. those exposed to antigen. –Upstream enhancer region: Binding sites for Ets-1, Gata-3, Lef, Nf- IL6. These proteins are only active in hematopoietic cells: helps to narrow the specificity of transcription.

15. Transcriptional control by viral trans- acting factors: Tat and Tar (Fig. 8.13). By itself, RNA Pol II does not elongate well away from the transcriptional control region: this provides a way to downregulate (negatively control) HIV replication. This is called poor processivity. To overcome this, the virus produces two trans- acting factors: –Tar: an RNA trans-acting factor –Tat: a protein trans-acting factor These two factors work synergistically to recruit other host proteins that stimulate and enhance the processivity of the elongating RNA Pol II complex.

16. Transcriptional control by viral trans-acting factors: Tat and Tar (Fig. 8.13).

17. DNA virus transcriptional programs Viral proteins act to either enhance or repress transcription of viral genes in an orderly manner.

18. DNA virus transcriptional programs SV40: Genome contains 2 transcriptional units: Early and Late. Early unit encodes the Large T antigen. Large T antigen acts to enhance transcription of the late unit, leading to virus production.

19. DNA virus transcriptional programs Adenovirus type 2: Has 3 transcriptional units: Immediate early, early, and late. E1A protein produced by immediate early unit E1A activates early unit, represses late unit. E2 protein encoded in early unit. E2 activates transcription of the late unit.