Protein-lipid interactions in influenza virus entry Peter Kasson - PowerPoint PPT Presentation

Protein-lipid interactions in influenza virus entry Peter Kasson Departments of Molecular Physiology and Biomedical Engineering University of Virginia Tuesday, May 12, 15

Why is it hard to predict pandemics 1000+ 900+ 800+ 700+ Cases 600+ 500+ 400+ 300+ 200+ 100+ 0+ 1995+ 2000+ 2005+ 2010+ 2015+ Year Sources: US CDC; Russell, Kasson et al., 2014 Tuesday, May 12, 15

b r ane o f ce l ls b y l o w e r i ng t he m ed i u m p H ( 5 , 19 , 54 ) . I f f o w l ob s e rv ed . I n con tr a st , f e rr i t i n w a s a t t ached t o t he p l a s m a p l ague vi r u s i n f ec ts M D C K ce l ls b y an endoc yt o t i c pa t h w a y m e m b r ane on ly i n s a m p l e s e x po s ed t o l o w p H ( F i g . 10 ) . I n pa ssi ng t h r ough t he lys o s o m e s, i t m i gh t a ls o be e x pec t ed t o s e v e r a l ca s e s, c l ea r con t i nu i ty be t w een t he ce l l and vi r u s m e m - f u s e a t t he p l a s m a m e m b r ane i f e x po s ed t o l o w p H . Th is b r ane s w a s ob s e rv ed , w i t h f e rr i t i n on ly bound t o t he p r o t r ud i ng s ee m ed e s pec i a l ly l i ke ly si nce l o w p H - dependen t he m o lysis vi r u s p r o f i l e ( F i g . l 0 a and b ) . Fu si on o f vi r u s e s t o m e m b r ane and ce l l - ce l l f u si on had been r ecen t ly de m on st r a t ed f o r i n f l u - v e si c l e s appa r en t ly s hed f r o m t he ce l ls w a s a ls o ob s e rv ed ( no t en z a vi r u s e s ( 19 , 55 , 56 ) . To t e st t h is, ce l ls w i t h p r ebound vi r u s s ho w n ) . L o w p H t r ea t m en t o f M D C K ce l ls i n t he ab s ence o f w e r e s u s pended i n m ed i a o f p H 5 . 0 and p H 7 . 4 f o r 1 m i n a t vi r u s p r oduced s o m e d ist u r bance o f t he p l a s m a m e m b r ane bu t 37 ° C and e x a m i ned b y t r an s m issi on e l ec t r on m i c r o s cop y a f t e r d i d no t i nduce a rt i f ac t ua l f e rr i t i n b i nd i ng . i nd i r ec t f e rr i t i n i mm uno l abe l i ng . I n t he ce l ls kep t a t p H 7 . 4 , To quan t i t a t e l o w p H - i nduced f u si on o f f o w l p l ague vi r u s f e rr i t i n w a s a ss oc i a t ed on ly w i t h vi r u s pa rt i c l e s and no t w i t h t o t he M D C K ce l l p l a s m a m e m b r ane , ce l ls w i t h p r ebound t he ce l l s u r f ace . N o f u si on o f t he vi r u s w i t h t he ce l l s u r f ace w a s r ad i oac t iv e vi r u s w e r e i ncuba t ed f o r 30 s a t 37 ° C w i t h m ed i a Cell entry by influenza Matlin et al., 1981 S t age s o f f o w l p l ague vi r u s en t ry i n t o M D C K ce l ls . C e l ls w i t h p r ebound vi r u s w e r e w a r m ed a t 37 ° C f o r d i f f e r en t t i m e s F I GUR E 8 and t hen f ix ed w i t h g l u t a r a l deh y de a t r oo m t e m pe r a t u r e . W i t h i n 5 m i n , vi r u s pa r t i c l e s w e r e s een i n s m oo t h s u r f aced p i ts and v e si c l e s ( a , b , and c ) , coa t ed p i ts ( d , e , and f) and coa t ed v e si c l e s ( g , h , and f, t he s a m p l e w a s st a i ned w i t h an t i - f o w l p l ague i ) . I n vi r u s s p i ke p r o t e i n I g G and t hen w i t h f e rr i t i n - goa t an t i - r abb i t I g G a f t e r f o r m a l deh y de f ix a t i on (s ee M a t e r i a ls and M e t hod s) . Th is i m age de m on st r a t e s t ha t pa r t o f t he vi r u s pa r t i c l e w a s t i gh t ly a ss oc i a t ed w i t h t he m e m b r ane si nce on l y t he e x po s ed pa r t is l abe l ed w i t h f e rr i t i n . A f t e r 10 m i n , vi r u s e s w e r e ob s e rv ed i n endo s o m e s ( j ) and m u l t iv e si cu l a r bod i e s ( k and I) . The i m age s s ho w n i n a , b , i n f a f t e r 1 m i n w a r m i ng , and i n j , k , and / a f t e r 10 m i n and c w e r e a f t e r 2 m i n w a r m i ng ; i n d , e , g , k , and i a f t e r 5 m i n w a r m i ng , w a r m i ng . a - i , x 62 , 500 ; k - l , x 50 , 000 . F I GUR E 10 Fu s i on o f f o w l p l ague vi r u s a t t he M D C K p l a s m a m e m b r ane . Fo w l p l ague vi r u s ( 60 j . g ) w a s bound t o M D C K ce l ls f o r Viral membrane 1 h a t 0 ° C and f u si on w a s i nduced b y i ncuba t i ng t he ce l ls f o r 1 m i n a t p H 5 . 0 and 37 ° C . The ce l ls w e r e t hen f ix ed w i t h f o r m a l deh y de and i mm uno l abe l ed w i t h an t i - f o w l p l ague vi r u s s p i ke I g G and f e r r i t i n - con j uga t ed goa t an t i - r abb i t I g G . V i r u s e s a r e c l ea r ly r ecogn i z ab l e b y t he f e rr i t i n a t t ached t o t he i r m e m b r ane . I n a t he nuc l eocap s i d is st i l l c l ea r ly visi b l e . I n b t he cap s i d is ba r e ly r ecogn i z ab l e , bu t t he vi r u s s hape and f e rr i t i n - l abe l ed s p i ke s a r e st i l l ob v i ou s . I n c t he s p i ke p r o t e i n s ha v e p r e s u m ab l y d i f f u s ed i n t he p l ane o f t he m e m b r ane a w a y f r o m t he si t e o f f u si on . A vi r u s p r o f i l e is st i l l de t ec t ab l e ( a r r o w ) . B a r 0 . 2 j m . x 106 , 000 . I n f l uen z a V i r u s En t ry 60 9 M A T U i v FT A t . Fusion Target and physical environment for fusion Target cell S t age s o f f o w l p l ague vi r u s en t ry i n t o M D C K ce l ls . C e l ls w i t h p r ebound vi r u s w e r e w a r m ed a t 37 ° C f o r d i f f e r en t t i m e s F I GUR E 8 and t hen f ix ed w i t h g l u t a r a l deh y de a t r oo m t e m pe r a t u r e . W i t h i n 5 m i n , vi r u s pa r t i c l e s w e r e s een i n s m oo t h s u r f aced p i ts and mutations block v e si c l e s ( a , b , and c ) , coa t ed p i ts ( d , e , and f) and coa t ed v e si c l e s ( g , h , and f, t he s a m p l e w a s st a i ned w i t h an t i - f o w l p l ague i ) . I n vi r u s s p i ke p r o t e i n I g G and t hen w i t h f e rr i t i n - goa t an t i - r abb i t I g G a f t e r f o r m a l deh y de f ix a t i on (s ee M a t e r i a l s and M e t hod s) . Th is i m age de m on st r a t e s t ha t pa r t o f t he vi r u s pa r t i c l e w a s t i gh t ly a ss oc i a t ed w i t h t he m e m b r ane si nce on l y t he e x po s ed pa r t is l abe l ed w i t h f e rr i t i n . A f t e r 10 m i n , vi r u s e s w e r e ob s e rv ed i n endo s o m e s ( j ) and m u l t iv e si cu l a r bod i e s ( k and I) . The i m age s s ho w n i n a , b , i n f a f t e r 1 m i n w a r m i ng , and i n j , k , and / a f t e r 10 m i n and c w e r e a f t e r 2 m i n w a r m i ng ; i n d , e , g , k , and i a f t e r 5 m i n w a r m i ng , w a r m i ng . a - i , x 62 , 500 ; k - l , x 50 , 000 . Tuesday, May 12, 15

Influenza fusion is heterogeneous on a single-virus level fusion e ffi ciency 10-40% depending on conditions in-cell fusion e ffi ciency ~10% even if we could simulate relevant timescales, a single movie wouldn’t do it 15 Num Fusion Events 10 5 Single fusion events detected via 0 0 50 100 150 200 fluorescence dequenching Waiting Time (s) Tuesday, May 12, 15

Bad combination Heterogeneous outcomes -> need many simulations for statistics Slow decorrelation times -> need long simulations Simplest full-scale systems >>1M particles -> need large simulations Biological system sensitive to fine details -> need high-fidelity simulations Tuesday, May 12, 15

Unraveling virus-membrane interactions surrounding fusion What are the relevant physical interactions controlling influenza viral fusion? Building an integrated understanding from simulations and biophysical experiments . Today: membrane interfaces preceding fusion, membrane-protein interactions. Tuesday, May 12, 15

Multi-pronged approach Building integrated understanding via statistical models at multiple levels “mid-scale” systems isolated components ~1-3M atoms 150K-800K atoms Tuesday, May 12, 15

Membranes form stable interfaces prior to fusion Depending on the system, these can be 10’s of ns to ~10 μ s Unexpected! Now good indirect experimental evidence! Tuesday, May 12, 15

Decreased water mobility at vesicle interface JACS 2011 Tuesday, May 12, 15

Glassy dynamics of water between two lipid membranes 20 with lipid diffusion correction 15 < t p > / < t x > 10 without correction 5 single bilayer lipids 0 0 10 5 15 N w /N l Implications for simulating fusion dynamics--can get stuck! Pronk, Lindahl, Kasson. JACS 2015 Tuesday, May 12, 15

Multi-level parallelism Need both parallelism at the individual “partition” level (MD scaling over N cores/GPU’s) and parallelism between partitions in solving the overall statistical problem Cluster Server Average: 0.04MB/s Initial sampling generate next Peak: 100MB/s SSL Latency: 10 ms Worker Worker Worker round parallel IB Average: 0.5GB/s simulations MPI MPI MPI Peak: >2.7GB/s Latency: 1-10 μ s Shared Average: 0.5GB/s model generation memory Thread Thread Thread Peak: 25GB/s Latency: <100ns SIMD SIMD SIMD SIMD SIMD SIMD SIMD SIMD SIMD SIMD SIMD SIMD Currently doing this ad-hoc on BW Copernicus: DAG engine to coordinate this Pronk et al., 2011; Pronk et al., 2015 Tuesday, May 12, 15

Hemagglutinin transmembrane domains • Hemagglutinin is trimeric; multiple trimers likely act together in fusion • Truncations to the TM domain can arrest fusion • Could TM-TM interactions play a role in fusion? statistical characterization many many simulations simulations many many starting conformational conditions samples • Multi-resolution approach to characterize TM interactions • Use coarse graining to sample di ff usional processes, atomistic simulations to sample conformational equilibria Tuesday, May 12, 15