Updated Proposal of Reference Sequences of HIV-1 Genetic Subtypes, 1997
1999 Nomencalture Proposal
Circulating Recombinant Forms
Thomas Leitner1, Bette Korber1, David Robertson2, Feng Gao3, Beatrice Hahn3
1Theoretical Biology and Biophysics, Group T-10, MS K710, Los Alamos National Laboratory, Los Alamos, NM 87545;
2 Laboratory of Structural & Genetic Information, CNRS-EP 91, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France;
3 Department of Medicine and Microbiology, University of Alabama at Birmingham, 701 S. 19th Street, LHRB 613, Birmingham, AL 35294.
HIV-1 subtypes are clusters of sequences within the M (major) group of HIV-1 viruses, that are defined by phylogenetic analysis 12. At the higher level HIV-1 is divided into group M (major) and group O (outlier) 9. In the 1996 compendium, a reference set of HIV-1 sequences representing the different subtypes was proposed. However, since that issue of the compendium, many more full length sequences have been produced from the various HIV-1 M group subtypes. Partly as a consequence of the availability of these new sequences, and partly due to further analysis of pre-existing data, new information has been accumulating concerning subtyping and HIV-1 alignments and hybrid genomes 3,6,8,14. We are thus updating the proposed list of reference sequences that appeared in section III, Table 1 on page 30 of the Human Retroviruses and AIDS 1996 compendium. In the future, we anticipate that this table, and the accompanying alignments, will be updated by the database on a regular basis as a service to the scientific community, particularly since new submissions are likely to include a considerable number of complex mosaic genomes.
Table 1 lists reference sequences for 9 group M subtypes in four major coding regions, i.e., gag, pol, env and nef. The criteria for inclusion of a reference sequence have changed since 1996, with a shift in emphasis to using full length HIV genomic sequences to serve as representatives for the subtypes, when such sequences are available. These are supplemented with sequences spanning intact coding regions, or else the longest gene fragments currently available. Table 1A summarizes a basic list of reference sequences alignments for international variation and subtyping efforts; while Table 1B provides additional information, including GenBank accession numbers, citations describing the respective sequence, sampling year, and the country where the virus was collected. Protein and nucleotide sequence alignments are available from the HIV database for each of the four coding regions on the website subtype alignments page. These alignments also contain the HIV-1 O group sequences ANT70 and MVP5180 and chimpanzee viral sequences CPZ-GAB and CPZ-ANT. Full length nucleotide genome alignments will also be available with the release of the next HIV compendium. The alignments were generated using first a hidden Markov model 4 at the nucleotide level, and then manually corrected at the amino acid level to keep open reading frames in frame, and finally back-translated into nucleotides. We are including maximum likelihood trees 5,15 showing the phylogenetic relationships of the representative sequences selected for the gag, pol, env and nef. Trees for V3 and p17 sequences are also included, because: 1) these genomic regions are commonly sequenced; 2) sequences are available for all of the selected reference strains, including the shorter fragments; and 3) the organization of the V3 region tree is somewhat altered relative to the full length env tree.
Some of the HIV-1 subtypes are more clearly defined than others. A, B, C, D, F, and H, each have at least one full length, apparently non-recombinant genome available as a reference sequence (non-recombinant in the sense that there are no conflicting subtype associations in different regions of the sequence identified at the present time), as well as multiple additional full length env and gag sequences. Our current database and methods, however, have limitations, and additional sequences and analyses in the future may change our current understanding of subtype relationships. Also, when using gene fragments, occasionally sequences may not give clear cut answers over short stretches. For example, the H full-length representative is essentially subtype H throughout its genome (see trees). However, phylogenetic analyses of V3 sequences on occasion produce trees that show a close association of subtype H and A sequences. Since this discordance is not always observed but depends on the particular alignments and programs used, it is not sufficient evidence for recombination. Nevertheless, the fact that it can occur, underscores the possbility of ambiguities that can arise with subtyping efforts, particularly those directed at short regions of the genome.
All full length representatives of subtypes E and G that have been sequenced to date represent mosaic genomes, with parts of the viral genome clustering with the A subtype in phylogenetic analysis, and parts of the genome forming the two clearly distinguishable clades designated either E or G. The E subtype clearly forms a distinct "E" subtype in envelope, appears to be an A/E mosaic in the HIV regulatory regions, and is essentially A-associated in gag and pol 3,8. All of the longer subtype G sequences available to date have stretches of subtype A-associated sequence interspersed. In contrast to the A/E mosaics, however, these A/G mosaic sequences have many different patterns of A-like sequence, suggesting that they resulted from independent recombination events 6,13,14[6, 13, 14]. Subtype I sequences [10] have also been reanalyzed 7,16 and there is now evidence from analysis of a complete genome that they are multiply mosaic and composed of 3 (or of possibly 4) subtypes 7. Further analysis is necessary to determine the potential they have to represent a distinct subtype. Only fragments of subtype J env and gag genes are available at this time 11, so further work is needed in this case as well to fully characterize these strains. However, these sequences are included as reference sequences to assist in identification of possible J subtype related strains in the future. In addition to the two subtype J sequences listed in the table, three other sequences (GM4, GM5, GM7) have been found to cluster close to this subtype over the env V3 region [1]. However, the sequence of GM4 was suggested to be a G/?/C recombinant 2, where the question mark covered a roughly 600 bp section including the V3 region.
Figure 3:
REFERENCES
1. Blouin, J. C., E. A. Guzman, and B. T. Foley. 1996. Global variation in the HIV-1 V3 region, p. III77-III201. In G. Myers and B. Korber and B. Foley and K.-T. Jeang and J. W. Mellors and S. Wain-Hobson (ed.), Human Retroviruses and AIDS: a compilation and analysis of nucleic and amino acid sequences. Los Alamos National Laboratory, Los Alamos, NM.
2. Bobkov, A., R. Cheingsong-Popov, M. Salminen, F. McCutchan, J. Louwagie, K. Ariyoshi, H. Whittle, and J. Weber. 1996. Complex mosaic structure of the partial envelope sequence from a Gambian HIV type 1 isolate. AIDS Res. Hum. Retrovirus. 12:169-171.
3. Carr, J. K., M. O. Salminen, C. Koch, D. Gotte, A. W. Artenstein, P. A. Hegerich, D. St. Louis, D. S. Burke, and F. E. McCutchan. 1996. Full-length sequence and mosaic structure of a human immunodeficiency virus type 1 isolate from Thailand. J. Virol. 70:5935-5943.
4. Eddy, S. 1995. HMMER Hidden Markov Models of Protein and DNA Sequence, 1.8 ed. Washington University School of Medicine, St. Louis, MO.
5. Felsenstein, J. 1993. PHYLIP: Phylogeny Inference Package, 3.52c ed. University of Washington, Seattle, WA.
6. Gao, F., D. L. Robertson, C. D. Carruthers, S. G. Morrison, B. Jian, Y. Chen, F. Barre- Sinoussi, M. Girard, A. Srinivasan, A. G. Abimiku, G. M. Shaw, P. M. Sharp, and B. H. Hahn. 1997. Non-recombinant reference clones and sequences for human immunodeficiency virus type 1 subtypes A, C, D, F, and H. :submitted.
7. Gao, F., D. L. Robertson, and B. H. Hahn. 1997. unpublished data.
8. Gao, F., D. L. Robertson, S. G. Morrison, H. Hui, S. Craig, J. Decker, P. N. Fultz, M. Girard, G. M. Shaw, B. H. Hahn, and P. M. Sharp. 1996. The heterosexual human immunodeficiency virus type 1 epidemic in Thailand is caused by an intersubtype (A/E) recombinant of African origin. J. Virol. 70:7013-7029.
9. Korber, B., I. Loussert-Ajakai, J. Blouin, and S. Saragosti. 1997. A comparison HIV-1 group M and group O functional and immunogenic domains in the gag p24 protein and the C2V3 region of the envelope protein, p. (III)41-(III)56. In G. Myers and B. Korber and B. Foley and K.-T. Jeang and J. W. Mellors and S. Wain-Hobson (ed.), Human retroviruses and AIDS 1996: a compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, NM.
10. Kostrikis, L. G., E. Bagdades, Y. Cao, L. Zhang, D. Dimitriou, and D. D. Ho. 1995. Genetic analysis of human immunodeficiency virus type 1 strains from patients in Cyprus: identification of a new subtype designated subtype I. J. Virol. 69: 6122-6130.
11. Leitner, T., A. Alaeus, S. Marquina, E. Lilja, K. Lidman, and J. Albert. 1995. Yet another subtype of HIV type 1? AIDS Res. Hum. Retrovirus. 11:995-997.
12. Louwagie, J., F. E. McCutchan, M. Peeters, T. P. Brennan, B. E. Sanders, G. A. Eddy, G. van der Groen, K. Fransen, G.-M. Gershy-Damet, R. Deleys, and D. S. Burke. 1993. Phylogenetic analysis of gag genes from 70 international HIV-1 isolates provides evidence for multiple genotypes. AIDS. 7: 769-780.
13. McCutchan, F. 1997. Personal communication.
14. McCutchan, F. E., M. O. Salminen, J. K. Carr, and D. S. Burke. 1996. HIV-1 genetic diversity. AIDS. 10 (suppl 3):S13-S20.
15. Olsen, G. J., H. Matsuda, R. Hagstrom, and R. Overbeek. 1994. fastDNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput. Appl. Biosci. 10: 41-48.
16. Salminen, M. 1996. Personal communication.
Table 1A.
Updated Proposal of Reference Sequences of HIV-1 Genetic Subtypes.
Subtype gag pol env nef
A U455 U455 U455 U455
92UG037.1 92UG037.1 92UG037.1 92UG037.1
K89 K89(KENYA)
VI32 SF170
B HXB2 HXB2 HXB2 HXB2
JRFL JRFL JRFL JRFL
OYI OYI OYI OYI
RF RF RF RF
C ETH2220 ETH2220 ETH2220 ETH2220
92BR025.8 92BR025.8 92BR025.8 92BR025.8
UG268 UG268
ZAM18 ZAM18
D NDK NDK NDK NDK
Z2Z6 Z2Z6 Z2Z6 Z2Z6
ELI ELI ELI ELI
94UG114.11 94UG114.11 94UG114.11 94UG114.11
E2 CM240
TN235
90CR402.1
93TH253.3
F 93BR020.1 93BR020.1 93BR020.1 93BR020.1
BZ162 BZ163
VI69 BZ126
VI174 RJI03
G3 92NG003.24 92NG003.24 92NG003.24
92NG083.1 92NG083.1 92NG083.1 92NG083.1
SE61655 SE61655 92UG975.10
92RU131.9
H 90CR056.1 90CR056.1 90CR056.1 90CR056.1
VI5575 VI5575
CA135
J SE70225 SE70225
SE78875 SE78875
1 The sequence 94UG114.1 is the most distant D subtype sequence (see trees),
tending to branch off closest to the B/D root in most analyses. In some subgenomic
regions, it may even move outside the B/D cluster.
2 E in most of env, A in gag and pol , mixture of A & E in regulatory genes [3, 8, 14].
3 The G reference sequences may show resemblance to subtype A in regions
of pol and vif, see also foot note 4.
4 92NG003.2 is a full length sequence, but is not included in the pol alignment
because of A-like regions in this gene [6].
5 Full length gene sequences of gag, pol or env are not yet available, see Table 1B.
Table 1B. Sequence descriptions
Subtype Sequence Acc. No. Source Region Sampling year Sampling country (origin)
HIV-1 M Group sequences in alignments
A U455 M62320 Oram, J.D. et al, ARHR 6:1073-1078 (1990) complete NA Uganda
A 92UG037.1 U51190 Gao, F. et al, J.virol 70:7013-7029 (1996) complete 1992 Uganda
A K89 L11774 Louwagie, J. et al, AIDS 7:769-780 (1993) gag NA Kenya
A VI32 L11788 Louwagie, J. et al, AIDS 7:769-780 (1993) gag NA Burundi
A SF170 M66533 Evans, L. et al, PNAS 85:2815 (1988) env NA Rwanda
B HXB2 K03455, M38432 Wong-staal, F. et al, Nature 313:277-284 (1985) complete NA France
B JRFL U63632 O'Brien, W.A. et al, Nature 348:69 (1990) complete NA US
B OYI M26727 Wain-Hobson, S. et al, AIDS 3:707 (1989) complete NA Gabon
B RF M17451, M12508 Starcich, B.R. et al, Cell 45:637-648 (1986) complete 1983 US (Haiti)
C ETH2220 U46016 Salminen, M.O. et al, ARHR 12:1329-1339 (1996) complete 1986 Ethopia
C 92BR025.8 U52953 Gao, F. et al, in prepapration (1997) complete 1992 Brazil
C UG268 L11799 Louwagie, J. et al, AIDS 7:769-780 (1993) gag 1993 Uganda
C UG268 L22948 Louwagie, J. et al, J.virol 69:263-271 (1995) env-nef 1993 Uganda
C ZAM18 L03705 McCutchan, F. et al,JAIDS 5:441-449 (1992) gag 1989 Zambia
C ZAM18 L22954 Louwagie, J. et al, J.virol 69:263-271 (1995) env 1989 Zambia
D NDK M27323 Spire, B. et al, Gene 81:275-284 (1989) complete NA Zaire
D Z2Z6 M22639 Theodore, T. et al, unpublished (1988) complete NA Zaire
D ELI K03454, X04414 Alizon, M. et al, Cell 46:63-74 (1986) complete NA Zaire
D 94UG114.1 U88824 Gao, F et al, in prepapration (1997) complete 1994 Uganda
E CM240 U54771 Carr, J.K. et al, J.virol 70:5935-5943 (1996) complete 1990 Thailand
E TN235 L03698 McCutchan, F.E. et al, ARHR 8:1887-1895 (1992) env NA Thailand
E 90CR402.1 U51188 Gao, F. et al, J.virol 70: 7013-7029 (1996) complete 1990 Central African Republic
E 93TH253.3 U51189 Gao, F. et al, J.virol 70: 7013-7029 (1996) complete 1993 Thailand
F BZ162 L11751 Louwagie, J. et al, AIDS 7:769-780 (1993) gag NA Brazil
F VI69 L11796 Louwagie, J. et al, AIDS 7:769-780 (1993) gag NA Belgium (Rwanda)
F VI174 L11782 Louwagie, J. et al, AIDS 7:769-780 (1993) gag NA Zaire
F BZ163 L22085 Louwagie, J. et al, ARHR 10:561-567 (1994) env-nef NA Brazil
F BZ126 L22082 Louwagie, J. et al, ARHR 10:561-567 (1994) env-nef NA Brazil
F 93BR020.1 AF005494 Gao, F. et al, in preparation (1997) complete 1993 Brazil
F RJI03 U08974 Sabino, E.C., et al., J.virol 68:6340-6346 (1994) partial env NA Brazil
G 92NG003.2 U88825 Gao, F. et al, in preparation (1997) complete 1992 Nigeria
G SE6165 L40752, L40761 Leitner, T. et al, Virology 209:136-146 (1995) p17, RT 1993 Sweden (Central Africa)
G 92NG083.1 U88826 Gao, F. et al, in preparation (1997) complete 1992 Nigeria
G 92UG975.10 U27426 Gao, F. et al, J.virol 70:1651-1657 (1996) env 1992 Uganda
G 92RU131.9 U30312 Gao, F. et al, J.virol 70:1651-1657 (1996) env-nef 1992 Russia
H 90CR056.1 AF005496 Gao, F. et al, in preparation (1997) complete 1990 Central African Republic
H VI557 U09666 Janssens, W. et al, ARHR 10:877-879 (1994) V3-V5 NA Zaire
H VI557 L11793 Louwagie, J. et al, AIDS 7:769-780 (1993) gag NA Zaire
H CA13 U09667 Janssens, W. et al, ARHR 10:877-879 (1994) V3-V5 NA Cameroon
J SE7022 L41177, L41179 Leitner, T. et al, ARHR 11:995-997 (1995) V3, p17 1993 Sweden (Zaire)
J SE7887 L41176, L41178 Leitner, T. et al, ARHR 11:995-997 (1995) V3, p17 1994 Sweden
Additional sequences available in alignments
O Group ANT70 L20587 Vanden Haesevelde, M. et al, JVI 68:1586-1596 (1994) complete NA Cameroon
O Group MVP5180 L20571 Gurtler, L. et al, JVI 68:1581-1585 (1994) complete 1991 Cameroon
Chimpanzee SIV-CPZANT U42720 Vanden Haesevelde, M. et al,Virology 221:346-350 (1996) complete 1986 Zaire
Chimpanzee SIV-CPZGAB X52154 Huet, T. et al, Nature 345:356-359 (1990) complete NA Gabon
Unrooted phylogenetic trees calculated with maximum likelihood methods 5,15. The alignments for the various subgenomic regions are derived from the complete genomic alignment, as presented in the HIV database 1997. Each of the alignments are available from the HIV database. All alignments were globally stripped for the generation of trees to 1398 sites for the gag gene; 2994 sites for the pol gene; 2250 sites for the env gene; 282 for the nef gene; 426 for the p17gag fragment and 213 sites for the env V3 fragment. All trees, except the nef tree, were constructed using the program DNAML under the F84 substitution model 5 where nucleotide frequencies were derived from the datasets and the transition/transversion parameter was set to 3.0 for the gag gene, 2.0 for the pol gene, 1.5 for the env gene, 3.0 for the p17 fragment and 1.42 for the V3 fragment. The nef gene tree was calculated with the programs fastDNAml and DNArates15, to allow for different substitution rates across sites. This proved to be important for the topology of this gene tree in resolving subtypes B and D. Although G subtype sequences in the trees shown cluster seperate from subtype A, they cluster in Subtype A in some subgene regions 6. Subtypes B and D are generally close to each other in all analyses, but with nef gene it may be difficult to completely resolve them from each other. All trees are drawn to the same scale, thereby indicating the relative information density in the different regions.