A new human immunodeficiency virus derived from gorillas

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1 A new human immunodeficiency virus derived from gorillas Jean-Christophe Plantier, Marie Leoz, Jonathan E Dickerson, Fabienne De Oliveira, François Cordonnier, Véronique Lemée, Florence Damond, David L Robertson and François Simon. SUPPLEMENTARY INFORMATION Supplementary Methods Supplementary Figure 1 Supplementary Figure 2 Patient s clinical and epidemiological data Serological analyses Virus isolation Molecular characterization Phylogenetic analyses Western blot pattern obtained with serum from patient RBF168, collected in 2005 CD4 + cell counts and plasma viral load (pvl) in samples obtained from March 2004 to February 2009 Supplementary Figure 3 Maximum likelihood phylogeny inferred from concatenated nucleotide alignments corresponding to the partial sequences available for SIVgorBQ664 Supplementary Figure 4 Supplementary Figure 5 Supplementary Table 1 Supplementary Table 2 Supplementary methods Alternative phylogenetic positions of RBF168 (i and ii) and representation of this relationship as a trichotomy (iii), with alternative cross-species transmission scenarios (a-c) Schematic representation of the PCR strategy used to determine the near-full-length genome (8,673 bp) of strain RBF168 Primers used unsuccessfully to amplify HIV-1 group O partial Pol and Gp41 regions Primers and PCR conditions used for successful near-fulllength genome amplification and sequencing of strain RBF168 Patient s clinical and epidemiological data 1

2 The patient was born in Cameroon and lived in various towns around Yaoundé, the capital. She had six children, all born before All were breastfed. She reported that two of them died of non-infectious causes and that none of the living children are HIVseropositive. Her husband died in Cameroon in 1984 from suspected stroke-related complications. No information was available on his HIV serostatus. The patient reported having sexual partners in Cameroon after her husband s death; no information on their HIV serostatus was available. HIV infection was initially diagnosed on her arrival in France in However, she reported weight loss in 2003 and had frequent febrile episodes before her arrival in France. She reported no sexual partners since arriving in France. Serological analyses We used the following EIA and rapid tests, as recommended by the manufacturers: Architect HIV Combo (Abbott), Murex HIV-1, 2, O (Abbott), Genscreen Ultra HIV Ag/Ac (Bio-Rad), HIV DUO Quick (biomérieux), Vikia HIV1/2 (biomérieux) and Determine HIV-1/2 (Inverness). We used the New Lav Blot I (Bio-Rad) assay as a confirmatory test. Virus isolation Strain RBF168 was isolated from the subject s plasma and PBMC on donor PBMC as previously described 1. P24 antigen was detected in the culture supernatant with the Vidas HIV P24 II assay (biomérieux) after as little as three days, and serial culture supernatant samples were strongly reactive. 2

3 Molecular characterization Two different plasma samples ( and ) were used for molecular characterization. - Group-specific RT-PCR: amplification of partial fragments of the Polymerase (pol) and Envelope (env) genes, used for resistance genotyping, was performed as previously described for group M 2, 3 and as follows for group O: 60 µl of RNA solution was extracted from 140 µl of plasma with the QIAmp Viral RNA Minikit (Qiagen) and 10 µl of the extract was used in a RT-PCR reaction (final volume 50 µl) with the SuperScript III One-Step RT-PCR System using Platinum Taq (Invitrogen), 20 pmol of each primer (Supplementary Table 1) and 1.5 mm MgSO4. We used a Perkin Elmer Gene Amp PCR System 9700 with the following cycling conditions: 50 C for 30 min, 93 C for 2 min, and 35 cycles of 94 C for 30 s, 55 C for 30 s, 68 C for 2 min, followed by a 10 min final extension step at 72 C. Two microliters of amplification product was used in a 50 µl nested PCR reaction with the HotStar Taq Master Mix (Qiagen), 20 pmol of each primer (Supplementary Table 1) and 1.5 mm MgCl2. The cycling conditions were 95 C for 15 min, 30 cycles at 94 C for 30 s, 55 C for 30 s and 72 C for 1 min, and a 10 min final extension step at 72 C. - Non-specific XL-RT-PCR: we amplified the near-full-length genome (8,673 bp) as follows: RNA was extracted and RT-PCR was performed as described above using 20 pmol of each primer (Supplementary Table 2) and 1.4 mm MgSO4. We used a Perkin Elmer Gene Amp PCR System 9700 with the following cycling conditions: 50 C for 60 min, 93 C for 2 min, 10 cycles of 93 C for 15 s, 50 C for 30 s, and 68 C for 10 min, 3

4 followed by 28 cycles at 93 C for 15 s, 50 C for 30 s, and 68 C for 10 min, with a 20 s increment at each cycle, followed by a 7 min final extension step at 68 C. We performed six overlapping nested PCR amplifications as follows (Supplementary Fig. 5, Supplementary Table 2): 2 µl of XL-RT-PCR product was used in a 50 µl nested PCR reaction with the HotStar Taq Master Mix (Qiagen), 20 pmol of each primer (Supplementary Table 2) and 1.5 mm MgCl2. The cycling conditions depended on the target fragment (Supplementary Table 2) and consisted of 95 C for 15 min followed by 35 or 45 cycles of (94 C for 30 s, 55 C or 50 C for 30 s, and 72 C for 2.5 min or 3.5 min) and a final extension step of 72 C for 10 min. PCR products were purified with the Nucleospin Extract II kit (Macherey Nagel) and directly sequenced with the CEQ Dye Terminator Cycle Sequencing with Quick Start kit (Beckman), in an automated capillary CEQ 8000 DNA sequencer (Beckman), using a set of 36 primers (Supplementary Table 2). Phylogenetic analyses The HIV-1 genome sequence alignment was downloaded from the Los Alamos Database ( and pruned to retain only appropriate reference sequences (see Fig. 1a). Available SIVgor sequences (CP684, CP2135 and CP2139) and concatenated fragments of BQ664, from Takehisa et al. 4, together with the RBF168 sequence, were integrated into these alignments using the CLUSTALX Profile Alignment option 5. The alignment was manually edited where appropriate, maintaining codon boundaries and all ambiguous and gap-containing sites were excluded. 4

5 Nucleotide maximum likelihood (ML) trees were constructed with PAUP* version 4.0b10 6, using a six-category discrete gamma distribution, as well as the HKY model and TBR branch swapping, and BIONJ start tree. For amino acid sequences, ML trees were constructed with PHYML v , using the WAG substitution model, a fourcategory discrete gamma distribution, and BIONJ start tree. One thousand ML bootstrap replicates were performed. MCMC Bayesian analysis, with MrBayes v ,9, was used to generate posterior probabilities for groupings in the amino acid phylogeny. The GTR model was used for nucleotide alignments and the WAG substitution model was used for amino acid alignments, both with gamma-distributed rates at sites. One million iterations were performed with trees sampled every 100 generations, the first 25% being discarded as burn-in. ML and Bayesian analysis converged on the same tree topology for the concatenated amino acid (Fig. 1a) and nucleotide sequence alignments (Supplementary Fig. 3); note that the phylogenetic position of RBF168 differed between the amino acid and nucleotide analyses. RBF168 clustered closest to SIVgor (with highly significant support from bootstrapping and Bayesian posterior probabilities) in the regions available for CP684, CP2135 and CP2139 (data not shown). Similarity plotting was performed with RAT 10. RBF168 was compared to HIV-1 groups M, N and O, SIVgor, SIVcpzPts, and SIVcpzPtt (see Fig. 1a for the strains used) and average sequence similarity was plotted for each, using a window size of 250 amino acids and increments of 100 amino acids. Given the different phylogenetic groupings of RBF168 with SIVgor (Fig. 1a versus Supplementary Fig. 3), TREE-PUZZLE was used to evaluate alternative tree topologies with the one-tailed KH and SH tests (Supplementary Fig. 4i versus ii) using 5

6 the WAG substitution model and a four-category discrete gamma distribution. This analysis indicated that neither of these phylogenetic positions was significantly more likely than the other. To further test between the two tree topologies (Supplementary Fig. 4i versus ii), ML trees (not shown) were constructed for each BQ664 region (A-D) as defined by Takehisa et al. 4 with 1,000 bootstrap replicates and MCMC Bayesian analysis (as above). A significant Bayesian support value for Region A (gag fragment) placed BQ664 as the basal lineage (posterior probability 1.0), while a significant Bayesian support value for region B (pol fragment) placed RBF168 as the basal lineage within the SIVgor radiation (posterior probability 1.0). The ML bootstraps for both outgroup possibilities (BQ664 and RBF168) in regions A and B were not significant (59.1% and 62.5% respectively). Both Region C (accessory gene fragment) and Region D (env fragment) also suggested RBF168 as the basal lineage, but without significant bootstrap and Bayesian support values (51.4%/0.91 and 43.5%/0.59 respectively). Similar results were obtained with nucleotide sequence regions. This lack of support for RBF168 being either basal to or within the SIVgor cluster (Fig. 1a) prevents us from distinguishing between the two alternative topologies (Supplementary Fig. 4i versus ii). These phylogenetic relationships imply that the relationship between these lineages (RBF168, SIVgorBQ664 and SIVgorCP684/CP2135/CP2139) is effectively a trichotomy (Supplementary Fig. 4iii), their divergence having occurred relatively closely in the gorilla population. Supplementary references 1. Simon, F. et al. Cellular and plasma viral load in patients infected with HIV-2. AIDS 7, (1993). 6

7 2. Plantier, J.C. et al. HIV-1 resistance genotyping on dried serum spots. AIDS 19, (2005). 3. Dachraoui, R. et al. Monitoring of HIV-1 resistance in Tunisia (North Africa) with a dried plasma spot strategy. J Acquir Immune Defic Syndr 47, (2008). 4. Takehisa, J. et al. Origin and biology of simian immunodeficiency virus in wildliving western gorillas. J Virol 83, (2009). 5. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, (1997). 6. D. Swofford, PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods) Version 4, Sinauer Associates, Sunderland, Massachusetts (2003). 7. Guindon S. & Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology. 52(5): (2003). 8. Ronquist, F. & Huelsenbeck, J.P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, (2003). 9. Huelsenbeck, J.P. & Ronquist, F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, (2001). 10. Etherington, G.J., Dicks, J. & Roberts, I.N. Recombination Analysis Tool (RAT): a program for the high-throughput detection of recombination. Bioinformatics 21, (2005). 11. Schmidt, H.A. et al. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18, (2002). 7

8 12. Roques, P. et al. Phylogenetic analysis of 49 newly derived HIV-1 group O strains: high viral diversity but no group M-like subtype structure. Virology 302, (2002). 8

9 Supplementary figures and tables Supplementary Figure 1: Western blot pattern obtained with serum from patient RBF168, collected in The figure shows the reactivities of the RBF168 sample, and of HIV-1 group M-positive and HIV-1 group O-positive controls. The molecular masses of HIV-1 glycoproteins (Gp) and proteins (P) are indicated in kda. RBF168 M O «P» Gp160 Gp120 P68 P55 P52 Gp41 P40 P32 P24 P18 9

10 Supplementary Figure 2. CD4 + cell counts and plasma viral load (pvl) in samples obtained from March 2004 to February The figure shows pvl values obtained with the in-house technique and two Abbott assays LCx HIV RNA Quantitative (sample ) and RealTime HIV1 (sample ). The group M- specific assays with undetectable pvl Amplicor Monitor v1.5, Roche (<2.3 log copies per ml) and Generic HIV charge virale, Biocentric (<2.3 log copies per ml) are shown in the box. Viral loads and CD4 + cell coun log copies RNA / ml Roche undetectable assay Biocentric undetectable assay CD4 / mm Sequential sample in-house VL Abbott VL CD4 10

11 Supplementary Figure 3. Maximum likelihood phylogeny inferred from concatenated nucleotide alignments corresponding to the partial sequences available for SIVgorBQ664 4 ; 3,135 nucleotide positions remained after removing gap-containing sites. The support values (indicated for key nodes only) in black above the branches are from 1,000 maximum likelihood bootstraps (shown as %), while posterior probabilities from nucleotide Bayesian analysis are shown in green below the branches (shown as proportions). 11

12 Supplementary Figure 4. Alternative phylogenetic positions of RBF168 (i and ii) and representation of this relationship as a trichotomy (iii), with alternative cross-species transmission scenarios (a-c) (Supplementary Methods online). Putative cross-species transmission events are represented by open circles for postulated SIVcpz cross-species transmission events and closed circles for SIVgor to human transmission events. Note that these alternative possible cross-species transmission histories are illustrative and not exhaustive. The group O and SIVgorCP684/CP2135/CP2139 clusters are shown collapsed. 12

13 Supplementary Figure 5. Schematic representation of the PCR strategy used to determine the near-full-length genome (8,673 bp) of strain RBF168. The positions of the six fragments generated by nested PCR are shown as open boxes below the representation of the HIV-1 genome. vif tat nef 5 LTR gag vpu rev 3 LTR pol vpr env A C E B D F 13

14 Table 1. Primers used unsuccessfully to amplify HIV-1 group O partial Pol and Gp41 regions. step genome region primer sequence (5'-3') fragment size (bp) R T- P C R Pol Prot O (U) TTCAAYTGTGGRAAAGAGGGAC 1,399 bp Pol OL1 (L) CTAATTCCTTGATAGATTTGACT nested PCR Protease Prot 4 (U) CAGCCCCACCRATGGAGG 453 bp Nouv L (L) CATTGTTTTACTTTTGGTCCAT nested PCR Reverse Transcriptase Pol OF1 (U) CAGTATTRGTGGGACCTACTCCTGTT 805 bp Pol OL2 (L) GGCTGTACTGTCCAYTTGTCTG A RT-PCR Env V3DURA (U) b ATTCCAATACACTATTGTGCTCCA 1,698 bp gp41ne3' (L) TAAGTTGCTCAAGAGGTGGTA nested PCR gp41 OFU (U) TAAAACCTTTTAGTGTRGCAC 675 bp 8393L (L) GTTGATATCCCTGCCTAATG a U: upper primer; L: lower primer b primers derived from reference 12 14

15 Table 2. Primers and PCR conditions used for successful near-full-length genome amplification and sequencing of strain RBF168. step genome region primer a sequence (5'-3') fragment size (bp) annealing ( C) elongation cycles RT-PCR nested PCR sequencing nearly full-length genome cycling conditions LTR152 (U) CTCAATAAAGCTTGCCTTGA 8, min 10 nefol (L) GAGTAAATTAACCCWTCCAGTCC min incr 20 s 28 A: Gag LTR152 (U) CTCAATAAAGCTTGCCTTGA min 30 s 35 gagoxl (L) AGCAATGTCACTTCCTGTTG B: Gag/Pol gagcamu2 (U) b GCATGGGTAAAGGCAGTA 3, min 30 s 35 HIV1POL3L (L) TCTACTTGTCCATGCATGGCTTCTCCTTTTA C: Pol RTOXL1U (U) CTCCAYCCAGACAARTGGAC 1, min 30 s 35 intoxl1l (L) CCYTCACCTTTCCACAGGAG D: accessory genes intoxl2u (U) AGAGAYCCTATYTGGAAAGGAC 1, min 30 s 45 revoxl2l (L) GCGTTACTTACTGCTYTGGTACA E: accessory genes/env revoxl3u (U) AAGAAGAAATGGATCCAGTAGA 1, min 30 s 45 envoxl3l (L) CAWATCCTGCTGGAGCACA F: Env revo6u (U) ATCTCCYATGGCAGGAAGAAG 3, min 30 s 35 nefol (L) GAGTAAATTAACCCWTCCAGTCC LTR152 (U) CTCAATAAAGCTTGCCTTGA LTR LTR5V (U) b AAAATCTCTAGCAGTGGCGCCCGAACAGG LTRO1L (L) TGTCTTGAGAGCTGGYTCTA gagcamu2 (U) b GCATGGGTAAAGGCAGTA Ugap1 (U) CATCCTGCTCCTGTAGGACC Gag gagoxl (L) AGCAATGTCACTTCCTGTTG gagou (U) ATGTACAGYCCAGTGAGCATC Lgap1 (L) AGCTTGTTCTGCTCTGAGGG prot1 (U) ATGTGGACAGGAAGGTCAC Ugap2 (U) CTATGTACAGAAACAAGTACAACC prot4 (U) CAGCCCCACCRATGGAGG Lgap2 (L) AAGCCTCACAAATATGCCCC Ugap3 (U) TGACAGTATTAGATGTGGGG polor3 (L) GGGTCTCATTGTTYACACTAGGAATAG Pol RTOXL1U (U) CTCCAYCCAGACAARTGGAC Lgap3 (L) CATTTACTGTCCAAGCTTCC RTO2U (U) GCCTCCCACACAAATGAKATAAG RTO1L (L) AATTCCCATTCWGGAATCCA into3u (U) ATAGAYCAGGCACARGAAGATC HIV1POL3L (L) TCTACTTGTCCATGCATGGCTTCTCCTTTTA intoxl2u (U) AGAGAYCCTATYTGGAAAGGAC intoxl1l (L) CCYTCACCTTTCCACAGGAG vifo4l (L) TGTCCCTACCTKRCTATGTCC accessory genes revoxl3u (U) AAGAAGAAATGGATCCAGTAGA revo6u (U) ATCTCCYATGGCAGGAAGAAG revoxl2l (L) GCGTTACTTACTGCTYTGGTACA envo6l (L) TTGTGMTGCCCAAATATTATG Ugap4 (U) GCACTCTTCTATAGAACAGACC envoxl3l (L) CAWATCCTGCTGGAGCACA Env Lgap4 (L) TATTGATACTGCATACACCC gp41ne5 (U) b TAAGTGCAGCAGGTAGCACTAT gp41j (L) b CAGGCGAGCTCGGAGYTGT Ugap5 (U) ACCTGTATCGTTACAGACCC Lgap5 (L) GTTAGGAGGTCCTTGATCCC Nef nefou (U) ACTCCTCARAACAATGCAG nefol (L) GAGTAAATTAACCCWTCCAGTCC a U: upper primer; L: lower primer b primers derived from reference 12 15