Retroviral RNA Processing and stability

Transcription

1 Retroviral RN Processing and stability m 7 gag pol env src Karen Beemon Johns Hopkins niversity m 7 env src m 7 src

2 Retroviruses hijack host cell gene expression machinery to generate progeny virions

3 Simple retroviruses use cis-acting RN sequences to express unspliced RN 1) Stability Element (RSE) (pol): Stabilize RN with long 3 TR 2) Negative Regulator of Splicing (NRS) (gag): suppresses splicing, promotes p (pseudo-5 SS:binds 1/11 & SRs), promotes rapid lymphomas 3) Direct. Repeat (DR) (3 TR): Exports unspliced RN (virion assembly) Needs Tap and Dbp5. NRS RSE gag pol env DR1 src DR2 Rous sarcoma virus RN

4 Deletions in gag affect RSV RN splicing and stability rrigo and Beemon MB 1988

5 SPLIIN: How is the correct ratio of spliced/ unspliced viral RNs maintained? 5 splice site efficient, 3 splice sites inefficient (RSV & HIV) Exonic splicing enhancers and silencers Negative regulator of splicing (NRS) sequence in gag 5 splice site decoy, 500 nts downstream of 5 splice site lso promotes p at 3 LTR SD S gag ESE S NRS SR proteins snrnps hnrnp H 1 snrnp u/uu 11 snrnp u/ u/cc NRS : spl. : 0

6 Bulged in NRS 5 helix promotes 1 snrnp binding u/uu abello-villegas et al. RN 2004 (Yun-Xing Wang, NI)

7 NRS-d3 RN makes aberrant 50S spliceosomal complex NRS d 3 - heparin 50S B + heparin 28S 18S iles & Beemon, MB 2005

8 NRS sequesters 3 SS in aberrant complex 11

9 5 Prp8 is critical to spliceosome function, cross-links to 5 SS RINER et al. RN 2005; 11:

10 5 Prp8 does not X-link to the NRS 5 splice site Site-specific label at +1/+2 of NRS decoy 5 splice site NRS p d hprp8 iles & Beemon, MB 2005

11 1. The NRS is a 5 splice site decoy that inhibits splicing and promotes polyadenylation Prp8 is misplaced. How do NRS mutant LVs act in vivo?

12 Mutation at 919 (+5) converts splicing suppressor to 5 SS

13 LV with NRS point mutation induces rapid-onset lymphomas Percent Survival WT Weeks SD S S NRS LV c- myb Polony et al JVI

14 Retroviral Stability Element (RSE) Prevents Nonsense-Mediated mrn Decay (NMD) of viral RN with long 3 TR

15 The Rous sarcoma virus unspliced RN must evade host translation quality control RSE m 7 gag pol env src Long mrn (>9 kb) nspliced mrn Polycistronic Long 3 TR after the first open reading frame Long half-life (9-20 hrs)

16 Deletions in gag affect RSV RN splicing and stability rrigo and Beemon MB 1988

17 RN termination codon in gag leads to instability of unspliced RN PT gag NT 7kb 3 TR Nonsense codon at nt WT S Barker and Beemon, MB 1994

18 deletion downstream of gag NT also destabilizes RN NT WT gag NT NT RSE Rous Stability Element (RSE) Nts deleted mock WT RSE experimental 100% 26% control Weil and Beemon, RN 2006

19 Stability element is dispensable when termination occurs near the poly() tail wt Mini Mini + RSE 400 nts ~650 nts - wt Mini Mini +RSE PT Experimental ontrol % of wt

20 Dominant negative pf1 stabilizes RN lacking the RSE Fraction WT WT pf1 Dominant Negative pf1 PT RSE WT Implicates nonsense-mediated mrn decay Weil and Beemon, RN 2006

21 Nonsense codons are usually recognized by downstream exon-junction complexes (EJ) in mammals

22 Exon-junction complex (EJ) is a heterogeneous collection of proteins mrnp Exon nts Exon2 Export Localization p15 TP P56 Nonsense-mediated mrn decay Y14 Magoh REF/LY pf1 pf3 pf2 eif4iii BTZ RNPS1 SRm160 Splicing ncharacterized

23 an the RSE stabilize premature termination within the gag gene? Ter forward Ter reverse Ter Ter Ter RSE forward Ter RSE reverse Ter Ter

24 The Stability Element stabilizes PT-bearing viral RN Relative RN level Ter Ter RSE For Ter RSE Rev WT actinomycin D (hrs) exp WT Ter (1924) Ter RSE for Ter RSE rev control

25 Is there a stability element downstream of other viral genes? TR insertion into gag RSE pol TR env TR gag pol env src Ter src TR WT RSE

26 Sequence found after HIV-1 gag termination codon stabilizes termination in RSV HIV genome vpr tat vpu rev nef gag pol vif?? rev env tat

27 erf 1 and 3 stabilize RSE RN, so perhaps RSE promotes termination Fraction WT WT PT RSE erf1 + erf Experimental ontrol Johanna Withers

28 RSE migrates anomalously in denaturing gels 5% acrylamide 10% acrylamide Jason Weil

29 RSE sediments as monomer in analytical ultracentrifuge 3.9 x 10-4 Equilibrium Velocity g(s * ) 1.9 x x Sedimentation oefficient bs 260 nm Radius Frictional Ratio = 1.7 Michael Hadjithomas Van Moudrianakis

30 SHPE chemistry (Selective 2 -hydroxyl acylation analyzed by primer extension) + * * * * * * * * M M7 1M7 Kevin Weeks, N

31 Structure of the RSE

32 Model: TR sequences downstream of gag NT promote proper termination and RN stability NMD PT Faux TR: NMD NT No NMD NT Proper Termination: No NMD

33 RSE inhibits NMD at gag TER

34 RSE inhibits NMD at gag TER Does it promote translation termination?

35 Nuclease footprinting of ribosomes on mrns indicate in vivo ribosome positions Nicholas Ingolia

36 Ribosome footprints reveal in vivo translation

37 HX treated 1 hr Ribosome Profile of RSV

38 Summary When RSE is deleted, RN undergoes NMD RSE can convert PT to normal stop codon RSE is a highly structured element HIV 3 TR can stabilize an RSV PT-may be common element in retroviruses or all mrns with long 3 TRs Mechanism? Johanna Withers Promote termination-interact with ribosome? Jason Weil Interact with Poly ()/PBP?