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

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). • elongatethrough 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) • Viral genome looks like a chromosome: -Associated with host-cell nucleosomes or viral proteins that look like nucleosomes. e.g. Papilloma- and Polyomaviruses • Viral ssDNA converted to dsDNA in viral particle. dsDNA then imported into nucleus. e.g. hepadnaviruses • 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.