basic research or basis for routine diagnostics? · nijhuis et al. 2007 cleavage site mutations...

AREVIR, April 2007, Elena Litau

University of CologneInstitute of Virology

Basic research or basis for routine diagnostics?

Gag cleavage site mutations:

Elena Litau



Background

Cleavage site mutations could compensate impaired enzymatic activity of mutant proteases.

Prabu-Jeyabalan et al. 2004

Cleavage site mutations alone confer protease inhibitor resistance by increased cleavage of gag precursor proteins.

Nijhuis et al. 2007

Cleavage site mutations occur in vitro and in vivo under selective pressure of protease inhibitors (PI).

Zhang et al. 1997, Doyon et al. 1996

Cleavage site mutations are associated with specific resistance profiles.

Verheyen et al. 2006

HIV can use an alternative mechanism to became resistent to PI - by changing the substrate instead of protease!



Analysed regions

PR/RT

C-terminal gag region (p7/p1 and p1/p6)

gag

C-terminal gag region and PR

and C-terminal gag region



Routine diagnostics

p6-2



>1194GATTGTACTGAGAGACAAGCTAATTTTTTAGGGAAAATCTGGCCTTCCCACAAGGGGAGGCCAGGGAATTTCCTTCAGAGCAGACCAGAGCCAACAGCCCCAACAGCCCCACCAGAAGAGAGCTTCAGGTTTGGGGAGGAGACAACAACTCCCTCTCAGAAG

Gag mutations:

431V, 436R, 437V, 449F, 449V, 451T, 453L, 453A, 453I

insertion: -

Subtype: B

Analysis



Q430 0 0,0% 4 1,7%K 0 0,0% 4 1,7%

A431 7 2,4% 88 36,4%V 5 1,7% 87 36,0%

K436 14 4,9% 30 12,4%R 10 3,5% 24 9,9%

I437 23 8,0% 45 18,6%V 11 3,8% 36 14,9%

428-437any therapy-associated

CS mutation24 8,4% 133 55,0%

CS p7/p1 mutations TN-viruses TE-virusesn=287 n=242

L449 31 10,8% 55 19,2%F 4 1,4% 28 9,8%V 2 0,7% 12 4,2%H 0 0,0% 4 1,4%

S451 66 23,0% 37 12,9%T 1 0,3% 9 3,1%

R452 6 2,1% 15 5,2%S 0 0,0% 6 2,1%

P453 34 11,8% 80 27,9%L 22 7,7% 57 19,9%A 0 0,0% 7 2,4%

443-453any therapy-associated

CS mutation28 9,8% 105 43,4%

n=287 n=242CS p1/p6-gag mutations TN-viruses TE-viruses

p7/p1 ERQAN - FLGKI p1/p6-gag RPGNF - LQSRP

CS mutations in TN (therapy-naive) and TE (therapy-experienced) viruses



CS mutations in different groups of therapy-naive and therapy-experienced viruses

13,5 16,7

33,3

83,375,0

100,0

40,0

63,3

86,9 87,593,8

0

20

40

60

80

100

without

prim

.Res

ista

nce

with R

Tm

uta

tions

1 m

ajor

PR

2 m

ajor

PR

3 m

ajor

PR

4 m

ajor

PR

1 m

ajor

PR

2 m

ajor

PR

3 m

ajor

PR

4 m

ajor

PR

5 m

ajor

PR

TN-HIV TE-HIV

Major- PR mutations:

24I, 32I, 46I/L, 50V/L,

54V/L/M/S/T/A, 82A/F/T/S, 84V, 90M

Per

cent

of C

S

posi

tive

HIV

n=277 n=242n=132



Covariance analysis in the group of therapy-experienced viruses

L449F

D30N M46L I54V

A431V

I84V V82A

P453AP453L



Cross-sectional data analysis of TE patients

p7/p1 ERQAN - FLGKI

p1/p6-gag RPGNF - LQSRP



Development of CS and PR mutations

Pat.: PH Subtyp B

Zeit 03/0205/98 07/02 05/03 03/04

Therapie d4T, 3TC, NVP LPV, AZT 3TC, TDF, ddI

Viruslast

PR-Mut.

CS-Mut.

77I

52929.400

449P, 453T

153.605

46I/M, 54V/I, 77I, 82F/V

431V, 449P, 453T/I

50.320

07/04

LPV/r, EFV

13.654 31.322

03/05

10I, 46I, 54V,77I, 82F

431V, 449P, 453I

10I, 46I, 54V,77I, 82F

431V, 449P, 453I

08/03

8.152

p7/p1 ERQAN - FLGKI

p1/p6-gag RPGNF - LQSRP



Conclusions

Several CS mutations are associated with therapy-exposure but can also be found in TN viruses. The clinical impact of CS mutations in these viruses has still to be determined.

CS mutations in TN and TE viruses are correlated with an increased number of primary PR mutations.

Therapy-associated CS mutations can be correlated with different resistance mutations (431V-24I/46I/L/54V/82A, 449F-30N, 453L-90M-84V) and integrated into predicted resistance pathways.

CS mutations are commonly involved in PI resistance and should be considered in HIV genotypic resistance tests.



Gag cleavage site mutations: basic research or basis for routine diagnostics?

Either!



Thank you

Jens Verheyen Institute of Virology, University of CologneMonika Timmen-WegoMelanie BalduinNadine Sichtig Dörte Hammerschmidt Martin DäumerRolf Kaiser

Ulrike Schuldenzucker caesar, Bonn

Daniel Hoffmann Center for Medical Biotechnology, University of Duisburg-Essen

Tobias Sing MPI for Informatics, SaarbrückenThomas Lengauer

Hauke Walter Institute of Clinical and Molecular Virology,Monika Tschochner German National Reference Center for Retroviruses,

University of Erlangen-Nürnberg

Mark Oette Dept. of Gastroenterology, University of DüsseldorfGerd Fätkenheuer Dept. of Internal Medicine I, University of CologneJürgen K. Rockstroh Dept. of Internal Medicine I, University of Bonn

basic research or basis for routine diagnostics? · nijhuis et al. 2007 cleavage site mutations...

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