THLBA105

Epistatic effect and positive selection in the HIV-1

vif

gene are linked with APOBEC3G/F neutralization activity

Élcio Leal 1 , Shiori Yabe 3 , Hirohisa Kishino 3 , Maria Clara Bizinoto 2, de Oliveira Martins 4 , Mariana Leão de Lima 2 , Edsel Renata Morais Ricardo Sobhie Diaz 2 , Luiz Mário Janini 2 Leonardo 2 , 1 2 Federal University of Pará, Belém, Brazil Federal University of São Paulo, São Paulo, Brazil 3 Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan 4 Bioinformatics and Molecular Evolution Laboratory, Department of Biochemistry, Genetics and Immunology, University of Vigo, Spain

APOBEC (

apolipoprotein B mRNA-editing catalytic polypeptide)

3G/F and vif

Innate Immunity

APOBEC 3G APOBEC 3F

C U G A Hypermutation vif TAA TGA = x TAG

Viral extinction

Proteasomal degration

To explore in more detail the host-virus evolutionary interaction at the population level, we evaluated the influence of A3G polymorphisms in the HIV-1 diversification and disease status.

Methods:

•400 Patients •CD4 + T cell counts and Viral load •7 SNPs A3G (2 PCR fragments of 6.5 and 8 kb •

vif

gene sequencing •Hypermutation detection in the integrase region (HA yellow into agarose gel)

Methods (cntd):

•vif phylogenetic inference •Population dynamics •Bayesian skyline plot method (

Ne τ)

•Bayesian Markov chain Monte Carlo coalescent framework •Recombination detection using Bayesian approach (biomc2) •Positive selection

dN/dS ( Yang, Z., 2007 Mol Biol Evol 24: 1586-1591

) •Co-evolving sites BGM (

Poon, et al., 2007, PLoS Comput Biol 3: e231

) •Vif sites versus CD4 levels (permutation test) •A3G SNPs versus viral phylogenies (Bayesian framework, BaTS)

Results:

•36% of samples were hypermutated (integras •80% B, 16% BF, 3% C, 1% F, 1 sample AG •mean diversity, measured by pairwise distances assuming the HKY model, was 0.0931

± 0.0006 for clade B, and 0.0493

± 0.001 for C (vif) •No single vif amino acid positions were significantly associated with the CD4+T counts.

•No A3G polymorphism correlation with vif genealogies

SNP -571 correlated with CD4+ T cell counts (Mann-Whitney test

p-value=0.0076)

C/G tend to have lower CD4+ T cell counts compared to C/C individuals

Results:

•Peak of

vif

recombination in sites that are essential to recognize and neutralizes 3G complexes. •Positively selected sites between APOBEC3 binding sites (WKSLVK and YRHHY) of the vif gene Positively selected sites within BC-Box and Cullin5-Box (region that binds cellular elongin B and C to form complexes that trigger the ubiquitination and proteasomal degradation of the A3G proteins) •

Co-evolving sites concentrated in a CTL region and within BC box

•Epistatic sites located in two distinct regions of the vif gene •Epistatic codons associated with CD4 levels

Results:

•Peak of

vif

recombination in sites that are essential to recognize and neutralizes 3G complexes •Positively selected sites between APOBEC3 binding sites (WKSLVK and YRHHY) of the vif gene •Positively selected sites within BC-Box and Cullin5-Box (region that binds cellular elongin B and C to form complexes that trigger the ubiquitination and proteasomal degradation of the A3G proteins) •

Co-evolving sites concentrated in a CTL region and within BC box

•Epistatic sites located in two distinct regions of the vif gene •Epistatic codons associated with CD4 levels

HIV-1 vif gene Consensus subtype B

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD

BC-BOX

QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ

Cullin-BOX

KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH CTL/CD8 epitope regions

HIV-1 vif gene ( Positively selected sites )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRK R RYSTQVDPDLAD

BC-BOX

QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALI K PKK I Q

Cullin-BOX

KPPLPSV R KLTEDRWNKPQKTKGHRGSHTMNGH CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

HIV-1 vif gene ( Co-evolving sites )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGERDW H LG Q GVSIEWRK R RYSTQVDPDLAD

BC-BOX

QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALI K PKK I Q

Cullin-BOX

KPPLPSV R KLTEDRWNKPQKTKGHRGSHTMNGH Co-evolving sites detected by BGM CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

HIV-1 vif gene ( Co-evolving sites )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGERDW H LG Q GV S IEWRK R RYSTQVDPDLAD

BC-BOX

QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALI K PKK I Q

Cullin-BOX

KPPLPSV R KLTEDRWNKPQKTKGHRGSHTMNGH Co-evolving sites detected by BGM CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

HIV-1 vif gene ( Co-evolving sites )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGERDW H LG Q GV S IEWRK R RYSTQVDPDLAD

BC-BOX

QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVG S LQYLALTALI K PKK I Q

Cullin-BOX

KPPLPSV R KLTEDRWNKPQKTKGHRGSHTMNGH Co-evolving sites detected by BGM CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

HIV-1 vif gene ( Epistatic sites x CD4 levels )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGER D W H LG Q GV S IEWRK R RYSTQVDPDLAD

BC-BOX

QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVG S LQYLALTAL I K PKK I Q

Cullin-BOX

KPPLPSV R KLTEDRWNKPQKTKGHRGSHTMNGH CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels

HIV-1 vif gene ( Epistatic sites x CD4 levels )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGER D W H LG Q G V S IEWRK R RYSTQVDPDLAD

BC-BOX

QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVG S LQYLALTAL I K PKK I Q

Cullin-BOX

KPPLPSV R KLTEDRWNKPQKTKGHRGSHTMNGH CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels

HIV-1 vif gene ( Epistatic sites x CD4 levels )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGER D W H LG Q G V S IEWRK R RYSTQVDP D LAD

BC-BOX

QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVG S LQYLALTAL I K P K K I Q

Cullin-BOX

KPPLPSV R KLTEDRWNKPQKTKGHRGSHTMNGH CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels

HIV-1 vif gene ( Epistatic sites x CD4 levels )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGER D W H LG Q G V S IEWRK R RYSTQVDP D LAD

BC-BOX

Q LIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVG S LQYLALTAL I K P K K I Q

Cullin-BOX

KPPLPSV R KLTEDRWNKPQKTKGHRGSHTMNGH CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels

HIV-1 vif gene ( Epistatic sites x CD4 levels )

(A3F) (A3G) (A3G)

MENRWQVMIVWQVDRMRIRTWKSLVKHHMY I S K KAK G W F YRHHYES T HPRI

(A3)

SSEVHIPLGDAKLVITTYWGLHTGER D W H LG Q G V S IEWRK R RYSTQVDP D LAD

BC-BOX

Q LIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVG S LQYLALTAL I K P K K I Q

Cullin-BOX

KPPLPSV R KLTEDRWN K PQKTKGHRGSHTMNGH CTL/CD8 epitope regions Positive selected sites  >1 and p<0.99

Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels

Conclusions:

Most of the adaptive evolution in the

vif

gene probably was to optimize A3G/F and cellular proteins (

i.e

., elongins) binding and recognition.

Acknowledgements: Laboratory work performed at the Retrovirology laboratory of Federal University of Sao Paulo Brazil This work was supported by FAPESP, (Foundation for the Support of Research in the State of Sao Paulo; grant no. 06/50109-5) and by the Japan Society for the Promotion of Science (SPS KAKENHI) Grant-in-Aid for Scientific Research (B) 19300094.