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, Leonardo de Oliveira Martins 4, Mariana Leão de Lima 2, Edsel Renata Morais 2, Ricardo Sobhie Diaz 2, Luiz Mário Janini 2 1 Federal University of Pará, Belém, Brazil 2 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 THLBA105

APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide) 3G/F and vif APOBEC 3G APOBEC 3F C U G A Hypermutation TAA TGA = x TAG Viral extinction vif Proteasomal degration Innate Immunity

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 (Neτ) Bayesian Markov chain Monte Carlo coalescent framework Recombination detection using Bayesian approach (biomc2) Positive selection dN/dS ( Yang, Z., 2007 Mol Biol Evol 24: ) 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 ± for clade B, and ±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

(A3F) (A3G) (A3) BC-BOX Cullin-BOX MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH CTL/CD8 epitope regions HIV-1 vif gene Consensus subtype B

(A3F) (A3G) (A3) BC-BOX Cullin-BOX MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions HIV-1 vif gene (Positively selected sites) (A3G)

(A3F) (A3G) (A3) BC-BOX Cullin-BOX Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions Co-evolving sites detected by BGM MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH (A3G) HIV-1 vif gene (Co-evolving sites)

(A3F) (A3G) (A3) BC-BOX Cullin-BOX Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions Co-evolving sites detected by BGM MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH (A3G) HIV-1 vif gene (Co-evolving sites)

(A3F) (A3G) (A3) BC-BOX Cullin-BOX Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions Co-evolving sites detected by BGM MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH (A3G) HIV-1 vif gene (Co-evolving sites)

(A3F) (A3G) (A3) BC-BOX Cullin-BOX Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH (A3G) HIV-1 vif gene (Epistatic sites x CD4 levels)

(A3F) (A3G) (A3) Cullin-BOX Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels BC-BOX MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH (A3G) HIV-1 vif gene (Epistatic sites x CD4 levels)

(A3F) (A3G) (A3) BC-BOX Cullin-BOX Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH (A3G) HIV-1 vif gene (Epistatic sites x CD4 levels)

(A3F) (A3G) (A3) BC-BOX Cullin-BOX Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels (A3G) MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH HIV-1 vif gene (Epistatic sites x CD4 levels)

(A3F) (A3G) (A3) BC-BOX Cullin-BOX Positive selected sites  >1 and p<0.99 CTL/CD8 epitope regions Co-evolving sites detected by BGM Epistatic sites associated with CD4+ T cell count levels MENRWQVMIVWQVDRMRIRTWKSLVKHHMYISKKAKGWFYRHHYESTHPRI SSEVHIPLGDAKLVITTYWGLHTGERDWHLGQGVSIEWRKRRYSTQVDPDLAD QLIHLYYFDCFSESAIRNAILGHIVRPRCEYQAGHNKVGSLQYLALTALIKPKKIQ KPPLPSVRKLTEDRWNKPQKTKGHRGSHTMNGH (A3G) HIV-1 vif gene (Epistatic sites x CD4 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/ ) and by the Japan Society for the Promotion of Science (SPS KAKENHI) Grant-in-Aid for Scientific Research (B)