JOURNAL OF VIROLOGY, Dec. 1991, p. 7061-7065 0022-538X/91/127061-05$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 65, No. 12
A Highly Divergent Simian Immunodeficiency Virus (SIVstm) Recovered from Stored Stump-Tailed Macaque Tissues ARIFA S.
KHAN,'*
TERESA A. GALVIN,' LINDA J. LOWENSTINE,2'3 MYRA B. JENNINGS,4 MURRAY B. GARDNER,4 AND CHARLES E. BUCKLER1 Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892,1 and Department of Pathology, School of Veterinary Medicine,2
California Regional Primate Research Center,3 and Department of Medical Pathology, School of Medicine,4 University of California, Davis, California 95616 Received 31 July 1991/Accepted 19 September 1991
We report here the results of molecular analysis of a simian immunodeficiency virus (designated SIVstm) which was isolated from a rhesus monkey inoculated with stored lymph node tissue of an Asian stump-tailed macaque. The latter monkey had died in 1977 during an epidemic of acquired immunodeficiency and lymphoma at the California Regional Primate Research Center (L. J. Lowenstine, N. W. Lerche, P. A. Marx, M. B. Gardner, and N. C. Pedersen, p. 174-176, in M. Girard and L. Valette, ed., Retroviruses of Human AIDS and RelatedAnimal Viruses, 1988). Nucleotide sequence analysis of the gag and env regions indicates that SIVstm is an ancient member of the SIV/human immunodeficiency virus type 2 group; it is quite divergent from known SIVs isolated from African sooty mangabeys as well as from Asian macaques. Furthermore, of all SIV strains described to date, SIVStm is the most closely related to human immunodeficiency virus type 2.
Large outbreaks of AIDS-like disease in macaques at various primate centers have been associated with natural infections of monkeys with type D simian AIDS retroviruses (10, 15). On the other hand, such epizootics are rare in the case of simian immunodeficiency viruses (SIVs). However, SIV was found to be the causal agent in an epidemic of immune suppression and lymphoma in stump-tailed macaques (Macaca arctoides) which occurred at the California Regional Primate Research Center (CRPRC) from 1976 to 1978 (13). Lymph node tissue from a CRPRC monkey which had died of lymphoma in 1977 was stored at -70°C. In 1986, a homogenate of this lymph node was inoculated into a rhesus macaque (MMU21685) which was seronegative for SIV. At 2 months postinoculation, the latter monkey seroconverted and subsequently developed an AIDS-related complex-like syndrome including lymphadenopathy, splenomegaly, hepatomegaly, diarrhea, and rash. Blood from this monkey was inoculated into a second rhesus macaque (MMU22579) which subsequently developed clinical manifestations of AIDS (12). SIV was isolated from infected peripheral blood mononuclear cells of monkeys MMU21685 and MMU22579 by cocultivation with stimulated human peripheral blood mononuclear cells. The viruses were designated SIVstm21685 and SIVstm22579, respectively. In this study, we examined the latter isolate, and we refer to it as SIVstm here. We have previously analyzed the long terminal repeat sequence of SIVstm (9) and shown that this virus is a novel member of the SIV/human immunodeficiency virus type 2 (HIV-2) group (6). To ascertain the evolutionary relationship of SIVstm to the other members of this group, we have now determined nucleotide sequences in two additional regions: the highly conserved gag region and the highly variable env region. Nucleotide sequences of SIVstm gag were deter*
mined with polymerase chain reaction (PCR)-amplified fragments from infected human peripheral blood lymphocyte (PBL) DNA prepared as previously described (9). One microgram of DNA was amplified in a Perkin-Elmer Cetus (PEC) DNA Thermal Cycler with PEC Amplitaq Taq DNA polymerase under conditions recommended by the manufacturer, except as indicated below. Two gag fragments which together encompassed all of the gag region except 12 bp at the 3' terminus were amplified. The 5' fragment, designated GAG1.3, was amplified under conditions recommended by PEC, except that 5 U of Taq polymerase was used. Twentyfive cycles were performed; each cycle used primer annealing steps at 55°C for 1 min and extensions at 72°C for 5 min. The primers were based on the SIVmac142 sequence (Los Alamos HIV Sequence Database, 1990) with restriction sites (underlined below) added for cloning. The primers used were as follows: Sall 5'-AGAGAGGTCGACAGAGTGAGAGACTCCTGAGT-3' (nucleotide [nt] 851 to 870) EcoRI
3'-AGAGAGGAATTCCCTCTTTCAAGGCTTCTG-5' (nt 2170 to 2153) The nucleotide numbers in the parentheses are for SIVmac142. The fragment was purified on a Sephacryl S300 cDNA spin column (Pharmacia) to remove primer-dimers, cleaved with EcoRI and Sall, and subsequently cloned into pBR322 DNA. Two clones designated GAG1.3-7 and GAG1 3-9, each containing a 1.3-kb insert, were obtained. The 3' gag fragment, designated GAG2.0, underwent 30 cycles of amplification using primer annealing steps at 55°C for 1 min and extensions at 72°C for 5 min. For cloning, one microliter from the 100-,ul reaction mix was amplified again for 30 cycles with the same primers; however, the annealing steps were at 50°C for 1 min, and extensions were at 72°C for 7
Corresponding author. 7061
7062
J. VIROL.
NOTES
min. The primers used were as follows: 5'-TCTCCAGCACTAGCAGGTAGA-3' (nt 551 to 571) 3'-TCCAAAGAGAGAATTGAGGT-5' (nt 2568 to 2548) The amplified fragment was purified on a Sephacryl S300 column (Pharmacia); the ends were filled in with T4 polynucleotide kinase and blunt-end ligated to PvuII-cleaved pBR322 vector DNA. Two clones, designated GAG20-2 and GAG2.0-7' were obtained. Of these, GAG2.07 contained the expected 2.0-kb insert, whereas GAG202 was truncated and contained a 1.0-kb insert. Three nucleotide sequences in the 5' gag region (encompassing nucleotides 29 to 1341 in the GenBank-EMBL data base) were determined by using DNAs designated GAG2 GAG1.3-7, and GAG1 Of these, the last two were identical
1) to a termination codon in GAG2 were obtained by using GAG2.07 and GAG20-2 DNAs (encompassing nt 1290 to 1757 [GenBank-EMBL data base]). Since the nucleotides at position 1195 in GAG1 and at position 1546 in GAG2.0-2 were also present in the SIV consensus sequence, sequences of these cloned DNAs were selected for the analysis presented here. The nucleotide sequence of the gag region of SIVstm was compared with those of known SIVs and HIVs. The results shown in Table 1 indicated that SIVstm had only 83 to 84% 0-7'
3-9
sequence
0-7'
3-9
to each other
except
at one
base (T
-*
C at nucleotide 1195)
which resulted in an amino acid change from leucine (L) in GAG139 (at position 323 in Fig. 1) to proline in GAG1 3-7. In the case of the 3' gag region, two sequences which differed in
one
base (G
->
A at nt
1546), resulting in
an
amino acid
change from tryptophan (W) at position 440 in GAG2
SIVstm SIVmac
0-2
homology with the African isolate SIVSmmH4 (iso-
lated from a sooty mangabey [16]) as well as with the Asian isolates SIVmne (isolated from a pig-tailed macaque [1]) and SIVmac142 and SIVmac251 (isolated from rhesus macaques [3]) (Table 1). On the other hand, SIVSmmH4 had 87 to 89% identity in the gag region with SIVmne and SIVmac isolates, whereas viruses of the SIVmne and SIVmac groups were 97 to 98% homologous to each other. Nucleotide sequence comparison of the gag regions of SIVs and HIV-2 isolates indicated that SIVstm was the SIV most closely related to the HIV-2 isolates; SIVstm had 74 to 79% homology to the HIV-2
(Fig.
MGARSSVLSGKKADELEKVRLRPGGKKKYM4LKHlVVWAANELDRFGLAESLLESKEGCQKILTVLE
65
..A
65
S. .A
65
K .R. .D
65
....................
....
SIVsmm
....
HIV-2
....N
N.
E.
.R.
......................
.I
R
..
DN.
I....
SIVstm
I. ..3EAKQVVKRHLVVETGTADKMPATSRPTAPPSG PLVPTGSENLKSLFNTVCVIWCIHAEEKVK CHTE
SIVmac
............ ................
.....
I.Q
SIVsmm
............ ................
.....
I.Q
HIV-2
SIVstm SIVmac
........D..
......................
ET.K
.KLAQ...
130
E.N
..A
130
pl6\/p28 RGGNYPVQQVGGNYVHLPLSPRTLNAWVKL VEE3KKFGAEVVSGFQALSEGCTPYDINQMLNCVGE D .I.. ..........I ........
.........
SIVsmm
....
.........
....
.........
130 130
S
R
........
........
195 195 195
HIV-2
KR .A
SIVstm
HQAAMQIIREIINEEAADWDVQHPQPGPLP ?AGC)LREPSGSDIAGTTSTVEEQIQWMHRQQNPIPV 260 S.D Y .259 L A. Q2Q . ...... .R. Y 260 RD D Y.P. S I.. ...D.R. V.. 260
SIVmac SIVsmm
HIV-2
.V.
........
........
D
195
..........
L..........
..................
......... ....
....................
....
.......
........
HIV-2
GNIYRRWIQLGLQKCVRMYNPVNILDIKQG 'PKE3PFQSYVDRFYKSLRAEQADPAVKNWMTQTLLI 325 .T.A T ....V .324 .T ....V .................. T... ......... 325 ...T....... .................. T... ......... 325
SIVstm
p28\/p2 p2\/p8 QNANPDCKLVLKGLGMNPTLEEMLTACQGV JGGP?GQKARLMAEALKEAFQPGPLPFAAAQQQGRR
SIVstm
SIVmac.
... ......................
SIVs.r.n
...
SIVmac
.........
....
..............
.............. I....
389 389
LA.V.I
KR.P.K ............... .....
SIVs.mm
............... ...
HIV-2
R K ............... ....
.............................. . ...-
LR.DQ
V.K.Q.K
MG.S.I
387
SIVsrun
p8\/pl pl\/p6 TVKCWNCGKEGHTAKQCKAPRRQGCWKCGK .PGHQMAKCPERQVGFLGFGPWGKKPRNFPMAQIPQ D..A.. ..L. S.R. PI .MD.V VH.. A ....L.M.. S.R. I .T.
HIV-2
AIRY ........ S.R.
SIVstm SIVmac
SIVstm SIVmac SIVsmm
HIV-2
.........
.........
.N.A.... .L.
390
VT.A..
454 454 455
452
TEDL 502 GLTPTAPPEMPTAPPVDPAADLLRSYMQLG ,KKQRESRKTPYKEV .V... ........D. .EK...... 495 ..Q D. V KN. .KM. .R N.ER 496 ..E. .ER.. Q. .R ..1I. AD. QQ.ER TEDLLHLEQRETPHREE 510 ....
.V
SIVstm
p6\ VHLNSLFG----
SIVmac
L.
SIVsmm
L.
EDQ*
HIV-2
L.
KDQ*
.....
....
GDQ*
FIG. 1. Comparison of gag protein sequences of SIVs and HIV-2. The predicted amino acid sequence of SIVstm gag was based on nucleotide sequences of PCR-amplified fragments from SIVstm-infected human PBL DNA. The primers were based upon the SIVmacl42 sequence (designated SIVmml42 in the Los Alamos HIV Sequence Database [1990]). Amplified fragments were purified and subsequently cloned into a vector DNA. The DNAs were sequenced by the dideoxynucleotide chain termination method using T7 DNA polymerase (United States Biochemicals). The predicted amino acid sequence of SIVstm was derived from the nucleotide sequences of GAG1.3-9 and GAG2.0-2 DNAs, which are representative of the SIVstm gag sequence. This sequence was aligned with sequences of SIVmac25l (SlVmac), SIVsmmH4 (SIVsmm), and HIV-2BEN (HIV-2) obtained from the Los Alamos HIV Sequence Database (1990). Dots below the SIVstm sequence indicate identical amino acids; dashes indicate uncloned sequences; gaps are introduced to maximize alignment. Locations of the proteins are indicated as in the Los Alamos HIV Sequence Database.
VOL. 65, 1991
NOTES
isolates, whereas the macaque and sooty mangabey isolates only 71 to 76% homologous to the HIV-2 isolates. Comparison of the predicted amino acid sequences in the gag regions indicated that the map order of SIVstm proteins was the same as for other SIVs and HIV-2 strains; however, the sequences were distinct (Fig. 1). Several amino acid substitutions were present in SIVstm with respect to the other SIV/HIV-2 sequences; some of these (16 of a total of 93 substitutions) occurred at highly conserved amino acids present in SIVmac, SIVsmm, and HIV-2. The p28 region contained the smallest proportion of substituted amino acids (11.3%). In contrast, large proportions of amino acid substitutions (30 to 34%) were seen in each of the carboxy-terminal proteins, p2, p8, and p6. Additionally, a single amino acid was deleted in the p8 sequence of SIVstm with respect to the other SIVs and HIV-2, and seven amino acids were inserted in the p6 region. This insertion in SIVstm (at positions 466 to 472) represented an imperfect duplication of amino acids 459 to 465 with two mismatches at amino acids 463 and 464. The relatedness seen in the gag regions of SIVstm and other lentiviruses of the SIV/HIV-2 group was confirmed by analysis of the env sequences. SIVstm env nucleotide sequences were determined from two PCR fragments amplified from EcoRI-digested SIVstm-infected human PBL DNA (1 ,ug). The primers were based on the SIVmacl42 sequence (Los Alamos HIV Sequence Database, 1990). The first fragment, designated ENV1.0, was amplified under PCR conditions recommended by PEC, except that Promega Taq DNA polymerase and 1Ox buffer were used. The buffer contained (final concentrations) 50 mM KCI, 10 mM TrisHCl (pH 9.0), 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, and 0.1% Triton X-100. Thirty cycles of amplification were performed with primer annealing steps at 55°C for 1 min and extensions at 72°C for 3 min. The primers used were as follows: were
KpnI
EcoRI
5'-GAGGGTACCGAA1TCATGTGGACAAATTGCAGAGGAGA-3' (nt 7790 to 7812) Sall SphI 3'-GAGGCATGCGTCGACGMGAGAACACTGGCCTATA-5' (nt 8799 to 8780)
The fragment was purified on a Sephacryl S300 column, cleaved with EcoRI and Sall, and cloned into the same sites in pSL1190 vector DNA (Pharmacia). Three clones, designated ENV1.0_4, ENV1 0o5, and ENV1.0-6, each containing a 1.0-kb insert, were sequenced. The second env fragment, designated ENV3', was amplified under PCR conditions recommended by PEC, except that a 0.5 ,uM concentration of each oligomer was used to decrease primer-dimer formation and Promega Taq polymerase and 10 x buffer were used. Thirty cycles of amplification were performed with primer annealing steps at 55°C for 1 min and extensions at 72°C for 3 min. The primers used were as follows: KpnI
EcoRI
5'-GAGGGTACCGAATTCAAGAGTAGCAATCTATGTAG-3' (nt 8731 to 8750) Sall SphI 3'-GAGGCATGCGTCGACGTAATTCTGCCAATCTGGAA-5' (nt 9570 to 9551)
The fragment was cleaved with EcoRI and SalI and cloned into the same sites in pUC19 vector DNA. Three clones, designated ENV3.6, ENV3.10, and ENV3.11, each containing a 0.8-kb insert, were obtained and sequenced. For each fragment, three cloned DNAs were sequenced; two of each
7063
(ENV10_5 and ENV1 0-6; ENV3.6 and ENV3.11) were identical and thus were used for analysis. Comparison of the nucleotide sequences in the env regions of SIVs and HIVs indicated that SIVstm had only 80 to 81% nucleotide sequence homology in the env region with the other Asian isolates, SIVmne, SIVmac142, and SIVmaC251, and was about 84% related to the African isolate SIVsmmH4 (Table 2). In contrast, SIVsmmH4 was 85 to 86% homologous to the Asian isolates in the env region. As in the case of the gag analysis, SIVstm was the closest SIV to HIV-2; it had 68 and 81% identity in the env region with HIV-2RoD and HIV-2BEN, respectively, whereas the other SIVs were 61 to 78% related to these HIV-2 isolates. Analysis of the predicted amino acid sequences of SIVstm and other SIVs and HIV-2 strains indicated several changes. The majority of different amino acids in SIVstm with respect to the other lentiviruses were clustered in five regions (designated 1 to 5 in Fig. 2); of these, clusters 1 and 2 corresponded to the previously identified variable regions V4 and V5 (2), respectively. The fifth cluster, located in the transmembrane region, encompasses sequences where an in-frame premature termination codon is present in several cloned SIV DNAs (7). SIVstm lacked this terminator, thus predicting a full-length gp4O. To determine phylogenetic relationships between SIVstm and other members of the SIV/HIV-2 group, minimumlength evolutionary trees were generated (18) on the basis of nucleotide variations in the gag and env regions. Similar branching orders of divergence were seen. The results of the gag analysis are shown in Fig. 3A. SIVs from a sooty mangabey and macaques were only 11 to 13% different from one another and 24 to 30% different from the HIV-2 isolates. On the other hand, SIVstm was quite divergent from the other SIVs (16 to 17% different) and was the most closely related SIV to the HIV-2 isolates (21 to 25% different). Because of similarities in structure, biology, and pathogenesis, SIVs and HIVs are thought to have originated from a common ancestor (5, 17). In fact, on the basis of close genetic relatedness, SIVs are believed to be the progenitors of HIV-2 strains (8). To further understand the relationship between SIVs and HIVs, we have estimated the times of divergence of different viruses of the SIV/HIV-2 group by using 0.5% per year as the fixation rate of nucleotide substitution in the gag region, on the basis of HIV-1 data (18). Our calculations show that SIVSmm and SIVmac diverged at least 15 years ago (around 1974), whereas SIVstm branched from the SIVsmm/SIVmac group about 30 years ago (around 1963 [Fig. 3B]). Accordingly, the divergence of SIV and HIV-2 must have occurred more than 30 years ago. TABLE 1. Percent nucleotide sequence homology in gag of SIV and HIV-2 isolates % gag homology witha:
Isolate
SIVsmmH4 SIVmne SIVmac142 SIVmac251
SIVstm SIVsmmH4 SIVmne SIVmac142 SIVmac25i HIV-2ROD 82.8
84.0 82.6 83.0 HIV-2ROD 74.1 HIV-2BEN 78.6
88.7 87.4 87.8 70.5 75.0
96.8 97.2 71.6 76.1
98.0 70.3 74.7
70.7 75.2
84.7
" Percent identity was calculated from the branch lengths indicated in Fig. 3. Percent difference = (sum of branch lengths/total number of sites examined) x 100. Nucleotide sequences used in the analysis were obtained from the Los Alamos HIV Sequence Database (1990).
J. VIROL.
NOTES
7064
Since Asian macaques have not been found to be infected with SIVs in the wild (14), it is believed that this group of monkeys has acquired SIVs by cross-species contamination from African monkeys (8). The possible origin of SIV in stump-tailed macaques is unknown. Historically, stumptailed monkeys first arrived at the CRPRC in 1964, in the same shipments from the same importer as sooty mangabeys (11). Furthermore, in 1967 and 1968, some stump-tailed macaques were housed at the CRPRC with sooty mangabeys later found to be SIV seropositive and with African green monkeys of unknown SIV antibody status. On the basis of sequence analysis of SIVStm, it is likely that stump-tailed macaques were infected in the early 1960s from a sooty mangabey source or another as yet unidentified source. Presumably, cross-contamination might have occurred in the holding facilities before dispersion to the primate center or at the primate center itself, either because of the proximity of the animals to each other or because of contaminated needles. An important biological feature of SIVstm is its involvement in the epidemic of immunodeficiency disease in the monkey colony at the CRPRC in 1976 to 1978, which resulted in a mortality of 75%. This is the largest known naturally occurring epidemic of an SIV-induced AIDS-like disease in macaques. Further studies of the horizontal transmission of SIVstm may render this virus isolate valuable in the study of nonhuman primates as a model for human AIDS and in the development of AIDS vaccines. Nucleotide sequence accession numbers. The nucleotide sequences of SIVstm gag and env have been deposited in the GenBank-EMBL data base under accession numbers X60667 and X60668, respectively.
TABLE 2. Percent nucleotide sequence homology in env of SIV and HIV-2 isolates % env homology witha:
Isolate
SIVstm SIVsmmH4 SIV.. SIVmacl42 SIVmac251 HIV-2ROD
SIVsmmH4
SIVmne
SIVmacl42 SIVmac25i
84.4 80.9
80.0 80.1 HIV-2ROD 68.2 HIV-2BEN 81.0
85.6 84.6 84.8 65.4 78.1
97.0 97.2 61.8 74.6
97.7 60.9 73.6
61.1 73.8
78.9
a Percent identity was calculated as indicated in Table 1, footnote a, from a minimum-length evolutionary tree constructed as described in the legend to Fig. 3. Nucleotide sequences used in the analysis were obtained from the Los Alamos HIV Sequence Database (1990).
Although these calculations for SIV are based on the nucleotide mutation rate for HIV-1, it is noteworthy that the calculated dates for SIVStm (1963 and 1977) are supported by the historical dates. In fact, the calculated date of SIVstm isolation, 1977, coincides with the date of storage of the infected tissue from which this virus was subsequently recovered in 1986 (discussed above). Furthermore, the calculated date of SIVstm origin, 1963, approximates the date of arrival of stump-tailed macaques into captivity and the first known exposure of these monkeys to a possible source of SIV infection (discussed below). These analyses indicate that SIVstm is the oldest SIV of the SIV/HIV-2 group reported to date; its sequence is most representative of an ancient SIV progenitor to the SIV/HIV-2 group of viruses. 1
SIVstm SIVmac SIVSmm HIV-2
/ CD4 BINDING DOMAIN KERQQKNYVPCHIRQVINTWHRVGKNVYL I .. K....... RDVTTQR ... HRR ...... I .. K....... DQKGGRWKQQ. ..Q.K ..... ..K.I .K.......
MWTNCRGEFLYCKMNWFLNWVE RSTLEMKF ...................... ...................... ............
N.T ...
.
.
65 60 65 50
2
SIVstm SIVmac
SIVSmm HIV-2
PPREGDLTCNSTVTSIIANID
AEVAELYRLELGDYKLVEITPIGLAPTNVKR
1299 ..........M D.. 1244 S.R. 12! 9 I...S. . .....I. . .DQR. 11!5
NNNETN L.....L DG.Q.S ............... .E ID. I RTH E.A.E ..... ...............
.
................
...
.....
3 cM120 \/ gp32/40
SIVstm SIVmac S IVsinm
YT S
HIV-2
.S
M.
17' 9
SIVstm SIVmac SIVSmm
RLTVWGTKNLQTRVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWPNDSLVPDWNNMTWQEWERKV
258 254 259 244
HIV-2
SIVstm SIVmac
SIVSmm HIV-2
SIVstm SIVmac SIVSmm HIV-2
...
SR' RGVFVLGFLGFLATAGSAMGAASLTLTAQSRTLLAGIVQQQQQLLDAVKRQQELL GG. V ..............................................
GA . l.
...
.T..
V..S.V......
.
.........
.
........
R.
S..V.
A.................A..T. D. D A. ET....N .Q. ........... V.... .K. KQ.
............................
.........................................
...........
4 DFLEANITQLLEEAQVQQEKNMYELQKLNSWDVFGNWFDLASWVRYIQYG YLVIGIVML ....
IA . .E.A
I.1.K.. IK .
.
................................
RY .
S.S
.I.
IY
.........
IL.
T. T .
....... LI.L.VIG..IV[ K. I.V...A I
19: 18 9
1944
323 319
324 309
5 /premature stop in mac VVQMLARLRKGYRPVFSSPPSY HQQILIHKGQEQPTKEGTEE I...K. .Q F.*THTQQDPAL..R..K.GD ......... .......... A.V ...
L.S.F ....
G.
..
P.
H
...
E.G
P
DRG
AN.
.
FIG. 2. Comparative sequence analysis of env of SIVs and HIV-2. The predicted amino acid sequence of SIVstm was based on nucleotide sequences of PCR fragments amplified from EcoRI-digested SlVstm-infected human PBL DNA which were subsequently cloned into a vector DNA. Two cloned DNAs, ENV1.0 5 and ENV3 6, represented the SIVstm env sequence. This sequence was aligned with those of SIVmac25l (SIVmac), SIVsmmH4 (SIVsmm), and HIV-2BEN (HIV-2) (obtained from the Los Alamos HIV Sequence Database [1990]). Amino acid 1 corresponds to the amino acid at position 187 in the SIV/HIV-2 env consensus sequence. The designations, symbols, and alignment are as described in the legend to Fig. 1. The regions of greatest amino acid heterogeneity are boxed. Clusters 1 and 2 correspond to variable regions V4 and V5 (2), respectively.
VOL. 65, 1991
NOTES
7065
B 14.0
HUMAN
_
(1963) 8.5
STUMP-TAILED MACAOUE I
_
t
I
SIVSMMH4
SIVSTM-C( 1977) 1986 12.5
( 1974)
SIVSMMH4
1986
SIVMAC251
1986
1
12
10.
SIVMNE 1986
SOOTY MANGABEY
11
_58 57
17
SIVMAC142
SIVMNE
RHESUS MACAQUE PIG-TAILED MACAQUE
551
SIVMND MANDRILL FIG. 3. Phylogenetic relationships of lentiviruses of the SIV/HIV-2 group. (A) A minimum-length evolutionary tree was constructed from variation in the gag nucleotide sequence by using the PAUP computer program (4) with the global branch-swapping option MULPARB. The total number of sites examined was 1,386. The minimum length of the tree was 1,644, and the consistency index was 0.663. The tree was rooted at the midpoint of the greatest patristic distance. The indicated branch lengths are proportional to the minimum number of single-nucleotide substitutions necessary to generate the observed variations. The length of the vertical lines is for clarity only. (B) The number of years of divergence from a common ancestor, indicated above the branches, was determined as a fraction of the total number of years of divergence between any two isolates (calculated by dividing the sum of the branch lengths indicated in panel A by a fixation rate of 0.5% per year, the rate determined for HIV-1 gag [18]). The dates in parentheses represent estimated dates based upon the calculations. The dates not in parentheses are the dates of virus isolations. In the case of SIVstm, the calculated date is different from the date of virus isolation and coincides with the date when the tissue was stored in the freezer.
(This research was performed by Teresa A. Galvin in partial fulfillment of the requirements for a Ph.D. in genetics from George Washington University, Washington, D.C.) We are indebted to G. Myers for evolutionary tree analyses and thank M. A. Martin for critical review of the manuscript, N. Lerche for historical information regarding the CRPRC monkeys, J. Silver for helpful discussions, and A. Buckler-White for oligomer synthesIs.
1.
2. 3.
4.
5. 6.
7. 8.
REFERENCES Benveniste, R. E., L. 0. Arthur, C.-C. Tsai, R. Sowder, T. D. Copeland, L. E. Henderson, and S. Oroszlan. 1986. Isolation of a lentivirus from a macaque with lymphoma: comparison with HTLV-IIU/LAV and other lentiviruses. J. Virol. 60:483-490. Burns, D. P. W., and R. C. Desrosiers. 1991. Selection of genetic variants of simian immunodeficiency virus in persistently infected rhesus monkeys. J. Virol. 65:1843-1854. Daniel, M. D., N. L. Letvin, N. W. King, M. Kannagi, P. K. Sehgal, R. D. Hunt, P. J. Kanki, M. Essex, and R. C. Desrosiers. 1985. Isolation of T-cell tropic HTLV-III-like retrovirus from macaques. Science 228:1201-1204. Day, W. H. E. 1983. Computationally difficult parsimony problems in phylogenetic systematics. J. Theor. Biol. 103:429-438. Desrosiers, R. C. 1990. The simian immunodeficiency viruses. Annu. Rev. Immunol. 8:557-578. Desrosiers, R. C. 1990. A finger on the missing link. Nature (London) 345:288-289. Hirsch, V. M., P. Edmondson, M. Murphey-Corb, B. Arbeille, P. R. Johnson, and J. I. Mullins. 1989. SIV adaption to human cells. Nature (London) 341:573-574. Hirsch, V. M., R. A. Olmsted, M. Murphey-Corb, R. H. Purcell, and P. R. Johnson. 1989. An African primate lentivirus (SIVsm) closely related to HIV-2. Nature (London) 339:389-392.
9. Khan, A. S., T. A. Galvin, M. B. Jennings, M. B. Gardner, and L. J. Lowenstine. 1991. SIV from stump-tailed macaque (SIVstm) is a divergent Asian isolate. J. Med. Primatol. 20:167-171. 10. Letvin, N. L., K. A. Eaton, W. T. Aldrich, P. K. Sehgal, B. J. Blake, S. F. Schlossman, N. W. King, and R. D. Hunt. 1983. Acquired immunodeficiency syndrome in a colony of macaque monkeys. Proc. Natl. Acad. Sci. USA 80:2718-2722. 11. Lowenstine, L. J. Unpublished data. 12. Lowenstine, L. J., et al. Submitted for publication. 13. Lowenstine, L. J., N. W. Lerche, P. A. Marx, M. B. Gardner, and N. C. Pedersen. 1988. An epizootic of simian AIDS caused by SIV in captive macaques in the 1970's, p. 174-176. In M. Girard and L. Valette (ed.), Retroviruses of human AIDS and related animal viruses. Pasteur Vaccines, Paris. 14. Lowenstine, L. J., N. C. Pedersen, J. Higgins, K. C. Pallis, A. Uyeda, P. Marx, N. W. Lerche, R. J. Munn, and M. B. Gardner. 1986. Seroepidemiologic survey of captive Old-World primates for antibodies to human and simian retroviruses, and isolation of a lentivirus from sooty mangabeys (Cercocebus atys). Int. J. Cancer 38:563-574. 15. Marx, P. A., D. H. Maul, K. G. Osborn, N. W. Lerche, P. Moody, L. J. Lowenstine, R. V. Henrickson, L. 0. Arthur, M. Gravell, W. T. London, J. L. Sever, J. A. Levy, R. J. Munn, and M. B. Gardner. 1984. Simian AIDS: isolation of a type D virus and disease transmission. Science 223:1083-1086. 16. Murphey-Corb, M., L. N. Martin, S. R. S. Rangan, G. B. Baskin, B. J. Gormus, R. H. Wolf, W. A. Andes, M. West, and R. C. Montelaro. 1986. Isolation of an HTLV-III-related retrovirus from macaques with simian AIDS and its possible origin in asymptomatic mangabeys. Nature (London) 321:435-437. 17. Schneider, J., and G. Hunsmann. 1988. Simian lentiviruses-the SIV group. AIDS 2:1-9. 18. Smith, T. F., A. Srinivasan, G. Schochetman, M. Marcus, and G. Myers. 1988. The phylogenetic history of immunodeficiency viruses. Nature (London) 333:573-575.
Vol. 65, No. 12
A Highly Divergent Simian Immunodeficiency Virus (SIVstm) Recovered from Stored Stump-Tailed Macaque Tissues ARIFA S.
KHAN,'*
TERESA A. GALVIN,' LINDA J. LOWENSTINE,2'3 MYRA B. JENNINGS,4 MURRAY B. GARDNER,4 AND CHARLES E. BUCKLER1 Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892,1 and Department of Pathology, School of Veterinary Medicine,2
California Regional Primate Research Center,3 and Department of Medical Pathology, School of Medicine,4 University of California, Davis, California 95616 Received 31 July 1991/Accepted 19 September 1991
We report here the results of molecular analysis of a simian immunodeficiency virus (designated SIVstm) which was isolated from a rhesus monkey inoculated with stored lymph node tissue of an Asian stump-tailed macaque. The latter monkey had died in 1977 during an epidemic of acquired immunodeficiency and lymphoma at the California Regional Primate Research Center (L. J. Lowenstine, N. W. Lerche, P. A. Marx, M. B. Gardner, and N. C. Pedersen, p. 174-176, in M. Girard and L. Valette, ed., Retroviruses of Human AIDS and RelatedAnimal Viruses, 1988). Nucleotide sequence analysis of the gag and env regions indicates that SIVstm is an ancient member of the SIV/human immunodeficiency virus type 2 group; it is quite divergent from known SIVs isolated from African sooty mangabeys as well as from Asian macaques. Furthermore, of all SIV strains described to date, SIVStm is the most closely related to human immunodeficiency virus type 2.
Large outbreaks of AIDS-like disease in macaques at various primate centers have been associated with natural infections of monkeys with type D simian AIDS retroviruses (10, 15). On the other hand, such epizootics are rare in the case of simian immunodeficiency viruses (SIVs). However, SIV was found to be the causal agent in an epidemic of immune suppression and lymphoma in stump-tailed macaques (Macaca arctoides) which occurred at the California Regional Primate Research Center (CRPRC) from 1976 to 1978 (13). Lymph node tissue from a CRPRC monkey which had died of lymphoma in 1977 was stored at -70°C. In 1986, a homogenate of this lymph node was inoculated into a rhesus macaque (MMU21685) which was seronegative for SIV. At 2 months postinoculation, the latter monkey seroconverted and subsequently developed an AIDS-related complex-like syndrome including lymphadenopathy, splenomegaly, hepatomegaly, diarrhea, and rash. Blood from this monkey was inoculated into a second rhesus macaque (MMU22579) which subsequently developed clinical manifestations of AIDS (12). SIV was isolated from infected peripheral blood mononuclear cells of monkeys MMU21685 and MMU22579 by cocultivation with stimulated human peripheral blood mononuclear cells. The viruses were designated SIVstm21685 and SIVstm22579, respectively. In this study, we examined the latter isolate, and we refer to it as SIVstm here. We have previously analyzed the long terminal repeat sequence of SIVstm (9) and shown that this virus is a novel member of the SIV/human immunodeficiency virus type 2 (HIV-2) group (6). To ascertain the evolutionary relationship of SIVstm to the other members of this group, we have now determined nucleotide sequences in two additional regions: the highly conserved gag region and the highly variable env region. Nucleotide sequences of SIVstm gag were deter*
mined with polymerase chain reaction (PCR)-amplified fragments from infected human peripheral blood lymphocyte (PBL) DNA prepared as previously described (9). One microgram of DNA was amplified in a Perkin-Elmer Cetus (PEC) DNA Thermal Cycler with PEC Amplitaq Taq DNA polymerase under conditions recommended by the manufacturer, except as indicated below. Two gag fragments which together encompassed all of the gag region except 12 bp at the 3' terminus were amplified. The 5' fragment, designated GAG1.3, was amplified under conditions recommended by PEC, except that 5 U of Taq polymerase was used. Twentyfive cycles were performed; each cycle used primer annealing steps at 55°C for 1 min and extensions at 72°C for 5 min. The primers were based on the SIVmac142 sequence (Los Alamos HIV Sequence Database, 1990) with restriction sites (underlined below) added for cloning. The primers used were as follows: Sall 5'-AGAGAGGTCGACAGAGTGAGAGACTCCTGAGT-3' (nucleotide [nt] 851 to 870) EcoRI
3'-AGAGAGGAATTCCCTCTTTCAAGGCTTCTG-5' (nt 2170 to 2153) The nucleotide numbers in the parentheses are for SIVmac142. The fragment was purified on a Sephacryl S300 cDNA spin column (Pharmacia) to remove primer-dimers, cleaved with EcoRI and Sall, and subsequently cloned into pBR322 DNA. Two clones designated GAG1.3-7 and GAG1 3-9, each containing a 1.3-kb insert, were obtained. The 3' gag fragment, designated GAG2.0, underwent 30 cycles of amplification using primer annealing steps at 55°C for 1 min and extensions at 72°C for 5 min. For cloning, one microliter from the 100-,ul reaction mix was amplified again for 30 cycles with the same primers; however, the annealing steps were at 50°C for 1 min, and extensions were at 72°C for 7
Corresponding author. 7061
7062
J. VIROL.
NOTES
min. The primers used were as follows: 5'-TCTCCAGCACTAGCAGGTAGA-3' (nt 551 to 571) 3'-TCCAAAGAGAGAATTGAGGT-5' (nt 2568 to 2548) The amplified fragment was purified on a Sephacryl S300 column (Pharmacia); the ends were filled in with T4 polynucleotide kinase and blunt-end ligated to PvuII-cleaved pBR322 vector DNA. Two clones, designated GAG20-2 and GAG2.0-7' were obtained. Of these, GAG2.07 contained the expected 2.0-kb insert, whereas GAG202 was truncated and contained a 1.0-kb insert. Three nucleotide sequences in the 5' gag region (encompassing nucleotides 29 to 1341 in the GenBank-EMBL data base) were determined by using DNAs designated GAG2 GAG1.3-7, and GAG1 Of these, the last two were identical
1) to a termination codon in GAG2 were obtained by using GAG2.07 and GAG20-2 DNAs (encompassing nt 1290 to 1757 [GenBank-EMBL data base]). Since the nucleotides at position 1195 in GAG1 and at position 1546 in GAG2.0-2 were also present in the SIV consensus sequence, sequences of these cloned DNAs were selected for the analysis presented here. The nucleotide sequence of the gag region of SIVstm was compared with those of known SIVs and HIVs. The results shown in Table 1 indicated that SIVstm had only 83 to 84% 0-7'
3-9
sequence
0-7'
3-9
to each other
except
at one
base (T
-*
C at nucleotide 1195)
which resulted in an amino acid change from leucine (L) in GAG139 (at position 323 in Fig. 1) to proline in GAG1 3-7. In the case of the 3' gag region, two sequences which differed in
one
base (G
->
A at nt
1546), resulting in
an
amino acid
change from tryptophan (W) at position 440 in GAG2
SIVstm SIVmac
0-2
homology with the African isolate SIVSmmH4 (iso-
lated from a sooty mangabey [16]) as well as with the Asian isolates SIVmne (isolated from a pig-tailed macaque [1]) and SIVmac142 and SIVmac251 (isolated from rhesus macaques [3]) (Table 1). On the other hand, SIVSmmH4 had 87 to 89% identity in the gag region with SIVmne and SIVmac isolates, whereas viruses of the SIVmne and SIVmac groups were 97 to 98% homologous to each other. Nucleotide sequence comparison of the gag regions of SIVs and HIV-2 isolates indicated that SIVstm was the SIV most closely related to the HIV-2 isolates; SIVstm had 74 to 79% homology to the HIV-2
(Fig.
MGARSSVLSGKKADELEKVRLRPGGKKKYM4LKHlVVWAANELDRFGLAESLLESKEGCQKILTVLE
65
..A
65
S. .A
65
K .R. .D
65
....................
....
SIVsmm
....
HIV-2
....N
N.
E.
.R.
......................
.I
R
..
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I....
SIVstm
I. ..3EAKQVVKRHLVVETGTADKMPATSRPTAPPSG PLVPTGSENLKSLFNTVCVIWCIHAEEKVK CHTE
SIVmac
............ ................
.....
I.Q
SIVsmm
............ ................
.....
I.Q
HIV-2
SIVstm SIVmac
........D..
......................
ET.K
.KLAQ...
130
E.N
..A
130
pl6\/p28 RGGNYPVQQVGGNYVHLPLSPRTLNAWVKL VEE3KKFGAEVVSGFQALSEGCTPYDINQMLNCVGE D .I.. ..........I ........
.........
SIVsmm
....
.........
....
.........
130 130
S
R
........
........
195 195 195
HIV-2
KR .A
SIVstm
HQAAMQIIREIINEEAADWDVQHPQPGPLP ?AGC)LREPSGSDIAGTTSTVEEQIQWMHRQQNPIPV 260 S.D Y .259 L A. Q2Q . ...... .R. Y 260 RD D Y.P. S I.. ...D.R. V.. 260
SIVmac SIVsmm
HIV-2
.V.
........
........
D
195
..........
L..........
..................
......... ....
....................
....
.......
........
HIV-2
GNIYRRWIQLGLQKCVRMYNPVNILDIKQG 'PKE3PFQSYVDRFYKSLRAEQADPAVKNWMTQTLLI 325 .T.A T ....V .324 .T ....V .................. T... ......... 325 ...T....... .................. T... ......... 325
SIVstm
p28\/p2 p2\/p8 QNANPDCKLVLKGLGMNPTLEEMLTACQGV JGGP?GQKARLMAEALKEAFQPGPLPFAAAQQQGRR
SIVstm
SIVmac.
... ......................
SIVs.r.n
...
SIVmac
.........
....
..............
.............. I....
389 389
LA.V.I
KR.P.K ............... .....
SIVs.mm
............... ...
HIV-2
R K ............... ....
.............................. . ...-
LR.DQ
V.K.Q.K
MG.S.I
387
SIVsrun
p8\/pl pl\/p6 TVKCWNCGKEGHTAKQCKAPRRQGCWKCGK .PGHQMAKCPERQVGFLGFGPWGKKPRNFPMAQIPQ D..A.. ..L. S.R. PI .MD.V VH.. A ....L.M.. S.R. I .T.
HIV-2
AIRY ........ S.R.
SIVstm SIVmac
SIVstm SIVmac SIVsmm
HIV-2
.........
.........
.N.A.... .L.
390
VT.A..
454 454 455
452
TEDL 502 GLTPTAPPEMPTAPPVDPAADLLRSYMQLG ,KKQRESRKTPYKEV .V... ........D. .EK...... 495 ..Q D. V KN. .KM. .R N.ER 496 ..E. .ER.. Q. .R ..1I. AD. QQ.ER TEDLLHLEQRETPHREE 510 ....
.V
SIVstm
p6\ VHLNSLFG----
SIVmac
L.
SIVsmm
L.
EDQ*
HIV-2
L.
KDQ*
.....
....
GDQ*
FIG. 1. Comparison of gag protein sequences of SIVs and HIV-2. The predicted amino acid sequence of SIVstm gag was based on nucleotide sequences of PCR-amplified fragments from SIVstm-infected human PBL DNA. The primers were based upon the SIVmacl42 sequence (designated SIVmml42 in the Los Alamos HIV Sequence Database [1990]). Amplified fragments were purified and subsequently cloned into a vector DNA. The DNAs were sequenced by the dideoxynucleotide chain termination method using T7 DNA polymerase (United States Biochemicals). The predicted amino acid sequence of SIVstm was derived from the nucleotide sequences of GAG1.3-9 and GAG2.0-2 DNAs, which are representative of the SIVstm gag sequence. This sequence was aligned with sequences of SIVmac25l (SlVmac), SIVsmmH4 (SIVsmm), and HIV-2BEN (HIV-2) obtained from the Los Alamos HIV Sequence Database (1990). Dots below the SIVstm sequence indicate identical amino acids; dashes indicate uncloned sequences; gaps are introduced to maximize alignment. Locations of the proteins are indicated as in the Los Alamos HIV Sequence Database.
VOL. 65, 1991
NOTES
isolates, whereas the macaque and sooty mangabey isolates only 71 to 76% homologous to the HIV-2 isolates. Comparison of the predicted amino acid sequences in the gag regions indicated that the map order of SIVstm proteins was the same as for other SIVs and HIV-2 strains; however, the sequences were distinct (Fig. 1). Several amino acid substitutions were present in SIVstm with respect to the other SIV/HIV-2 sequences; some of these (16 of a total of 93 substitutions) occurred at highly conserved amino acids present in SIVmac, SIVsmm, and HIV-2. The p28 region contained the smallest proportion of substituted amino acids (11.3%). In contrast, large proportions of amino acid substitutions (30 to 34%) were seen in each of the carboxy-terminal proteins, p2, p8, and p6. Additionally, a single amino acid was deleted in the p8 sequence of SIVstm with respect to the other SIVs and HIV-2, and seven amino acids were inserted in the p6 region. This insertion in SIVstm (at positions 466 to 472) represented an imperfect duplication of amino acids 459 to 465 with two mismatches at amino acids 463 and 464. The relatedness seen in the gag regions of SIVstm and other lentiviruses of the SIV/HIV-2 group was confirmed by analysis of the env sequences. SIVstm env nucleotide sequences were determined from two PCR fragments amplified from EcoRI-digested SIVstm-infected human PBL DNA (1 ,ug). The primers were based on the SIVmacl42 sequence (Los Alamos HIV Sequence Database, 1990). The first fragment, designated ENV1.0, was amplified under PCR conditions recommended by PEC, except that Promega Taq DNA polymerase and 1Ox buffer were used. The buffer contained (final concentrations) 50 mM KCI, 10 mM TrisHCl (pH 9.0), 1.5 mM MgCl2, 0.01% (wt/vol) gelatin, and 0.1% Triton X-100. Thirty cycles of amplification were performed with primer annealing steps at 55°C for 1 min and extensions at 72°C for 3 min. The primers used were as follows: were
KpnI
EcoRI
5'-GAGGGTACCGAA1TCATGTGGACAAATTGCAGAGGAGA-3' (nt 7790 to 7812) Sall SphI 3'-GAGGCATGCGTCGACGMGAGAACACTGGCCTATA-5' (nt 8799 to 8780)
The fragment was purified on a Sephacryl S300 column, cleaved with EcoRI and Sall, and cloned into the same sites in pSL1190 vector DNA (Pharmacia). Three clones, designated ENV1.0_4, ENV1 0o5, and ENV1.0-6, each containing a 1.0-kb insert, were sequenced. The second env fragment, designated ENV3', was amplified under PCR conditions recommended by PEC, except that a 0.5 ,uM concentration of each oligomer was used to decrease primer-dimer formation and Promega Taq polymerase and 10 x buffer were used. Thirty cycles of amplification were performed with primer annealing steps at 55°C for 1 min and extensions at 72°C for 3 min. The primers used were as follows: KpnI
EcoRI
5'-GAGGGTACCGAATTCAAGAGTAGCAATCTATGTAG-3' (nt 8731 to 8750) Sall SphI 3'-GAGGCATGCGTCGACGTAATTCTGCCAATCTGGAA-5' (nt 9570 to 9551)
The fragment was cleaved with EcoRI and SalI and cloned into the same sites in pUC19 vector DNA. Three clones, designated ENV3.6, ENV3.10, and ENV3.11, each containing a 0.8-kb insert, were obtained and sequenced. For each fragment, three cloned DNAs were sequenced; two of each
7063
(ENV10_5 and ENV1 0-6; ENV3.6 and ENV3.11) were identical and thus were used for analysis. Comparison of the nucleotide sequences in the env regions of SIVs and HIVs indicated that SIVstm had only 80 to 81% nucleotide sequence homology in the env region with the other Asian isolates, SIVmne, SIVmac142, and SIVmaC251, and was about 84% related to the African isolate SIVsmmH4 (Table 2). In contrast, SIVsmmH4 was 85 to 86% homologous to the Asian isolates in the env region. As in the case of the gag analysis, SIVstm was the closest SIV to HIV-2; it had 68 and 81% identity in the env region with HIV-2RoD and HIV-2BEN, respectively, whereas the other SIVs were 61 to 78% related to these HIV-2 isolates. Analysis of the predicted amino acid sequences of SIVstm and other SIVs and HIV-2 strains indicated several changes. The majority of different amino acids in SIVstm with respect to the other lentiviruses were clustered in five regions (designated 1 to 5 in Fig. 2); of these, clusters 1 and 2 corresponded to the previously identified variable regions V4 and V5 (2), respectively. The fifth cluster, located in the transmembrane region, encompasses sequences where an in-frame premature termination codon is present in several cloned SIV DNAs (7). SIVstm lacked this terminator, thus predicting a full-length gp4O. To determine phylogenetic relationships between SIVstm and other members of the SIV/HIV-2 group, minimumlength evolutionary trees were generated (18) on the basis of nucleotide variations in the gag and env regions. Similar branching orders of divergence were seen. The results of the gag analysis are shown in Fig. 3A. SIVs from a sooty mangabey and macaques were only 11 to 13% different from one another and 24 to 30% different from the HIV-2 isolates. On the other hand, SIVstm was quite divergent from the other SIVs (16 to 17% different) and was the most closely related SIV to the HIV-2 isolates (21 to 25% different). Because of similarities in structure, biology, and pathogenesis, SIVs and HIVs are thought to have originated from a common ancestor (5, 17). In fact, on the basis of close genetic relatedness, SIVs are believed to be the progenitors of HIV-2 strains (8). To further understand the relationship between SIVs and HIVs, we have estimated the times of divergence of different viruses of the SIV/HIV-2 group by using 0.5% per year as the fixation rate of nucleotide substitution in the gag region, on the basis of HIV-1 data (18). Our calculations show that SIVSmm and SIVmac diverged at least 15 years ago (around 1974), whereas SIVstm branched from the SIVsmm/SIVmac group about 30 years ago (around 1963 [Fig. 3B]). Accordingly, the divergence of SIV and HIV-2 must have occurred more than 30 years ago. TABLE 1. Percent nucleotide sequence homology in gag of SIV and HIV-2 isolates % gag homology witha:
Isolate
SIVsmmH4 SIVmne SIVmac142 SIVmac251
SIVstm SIVsmmH4 SIVmne SIVmac142 SIVmac25i HIV-2ROD 82.8
84.0 82.6 83.0 HIV-2ROD 74.1 HIV-2BEN 78.6
88.7 87.4 87.8 70.5 75.0
96.8 97.2 71.6 76.1
98.0 70.3 74.7
70.7 75.2
84.7
" Percent identity was calculated from the branch lengths indicated in Fig. 3. Percent difference = (sum of branch lengths/total number of sites examined) x 100. Nucleotide sequences used in the analysis were obtained from the Los Alamos HIV Sequence Database (1990).
J. VIROL.
NOTES
7064
Since Asian macaques have not been found to be infected with SIVs in the wild (14), it is believed that this group of monkeys has acquired SIVs by cross-species contamination from African monkeys (8). The possible origin of SIV in stump-tailed macaques is unknown. Historically, stumptailed monkeys first arrived at the CRPRC in 1964, in the same shipments from the same importer as sooty mangabeys (11). Furthermore, in 1967 and 1968, some stump-tailed macaques were housed at the CRPRC with sooty mangabeys later found to be SIV seropositive and with African green monkeys of unknown SIV antibody status. On the basis of sequence analysis of SIVStm, it is likely that stump-tailed macaques were infected in the early 1960s from a sooty mangabey source or another as yet unidentified source. Presumably, cross-contamination might have occurred in the holding facilities before dispersion to the primate center or at the primate center itself, either because of the proximity of the animals to each other or because of contaminated needles. An important biological feature of SIVstm is its involvement in the epidemic of immunodeficiency disease in the monkey colony at the CRPRC in 1976 to 1978, which resulted in a mortality of 75%. This is the largest known naturally occurring epidemic of an SIV-induced AIDS-like disease in macaques. Further studies of the horizontal transmission of SIVstm may render this virus isolate valuable in the study of nonhuman primates as a model for human AIDS and in the development of AIDS vaccines. Nucleotide sequence accession numbers. The nucleotide sequences of SIVstm gag and env have been deposited in the GenBank-EMBL data base under accession numbers X60667 and X60668, respectively.
TABLE 2. Percent nucleotide sequence homology in env of SIV and HIV-2 isolates % env homology witha:
Isolate
SIVstm SIVsmmH4 SIV.. SIVmacl42 SIVmac251 HIV-2ROD
SIVsmmH4
SIVmne
SIVmacl42 SIVmac25i
84.4 80.9
80.0 80.1 HIV-2ROD 68.2 HIV-2BEN 81.0
85.6 84.6 84.8 65.4 78.1
97.0 97.2 61.8 74.6
97.7 60.9 73.6
61.1 73.8
78.9
a Percent identity was calculated as indicated in Table 1, footnote a, from a minimum-length evolutionary tree constructed as described in the legend to Fig. 3. Nucleotide sequences used in the analysis were obtained from the Los Alamos HIV Sequence Database (1990).
Although these calculations for SIV are based on the nucleotide mutation rate for HIV-1, it is noteworthy that the calculated dates for SIVStm (1963 and 1977) are supported by the historical dates. In fact, the calculated date of SIVstm isolation, 1977, coincides with the date of storage of the infected tissue from which this virus was subsequently recovered in 1986 (discussed above). Furthermore, the calculated date of SIVstm origin, 1963, approximates the date of arrival of stump-tailed macaques into captivity and the first known exposure of these monkeys to a possible source of SIV infection (discussed below). These analyses indicate that SIVstm is the oldest SIV of the SIV/HIV-2 group reported to date; its sequence is most representative of an ancient SIV progenitor to the SIV/HIV-2 group of viruses. 1
SIVstm SIVmac SIVSmm HIV-2
/ CD4 BINDING DOMAIN KERQQKNYVPCHIRQVINTWHRVGKNVYL I .. K....... RDVTTQR ... HRR ...... I .. K....... DQKGGRWKQQ. ..Q.K ..... ..K.I .K.......
MWTNCRGEFLYCKMNWFLNWVE RSTLEMKF ...................... ...................... ............
N.T ...
.
.
65 60 65 50
2
SIVstm SIVmac
SIVSmm HIV-2
PPREGDLTCNSTVTSIIANID
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1299 ..........M D.. 1244 S.R. 12! 9 I...S. . .....I. . .DQR. 11!5
NNNETN L.....L DG.Q.S ............... .E ID. I RTH E.A.E ..... ...............
.
................
...
.....
3 cM120 \/ gp32/40
SIVstm SIVmac S IVsinm
YT S
HIV-2
.S
M.
17' 9
SIVstm SIVmac SIVSmm
RLTVWGTKNLQTRVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWPNDSLVPDWNNMTWQEWERKV
258 254 259 244
HIV-2
SIVstm SIVmac
SIVSmm HIV-2
SIVstm SIVmac SIVSmm HIV-2
...
SR' RGVFVLGFLGFLATAGSAMGAASLTLTAQSRTLLAGIVQQQQQLLDAVKRQQELL GG. V ..............................................
GA . l.
...
.T..
V..S.V......
.
.........
.
........
R.
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A.................A..T. D. D A. ET....N .Q. ........... V.... .K. KQ.
............................
.........................................
...........
4 DFLEANITQLLEEAQVQQEKNMYELQKLNSWDVFGNWFDLASWVRYIQYG YLVIGIVML ....
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.
................................
RY .
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.I.
IY
.........
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T. T .
....... LI.L.VIG..IV[ K. I.V...A I
19: 18 9
1944
323 319
324 309
5 /premature stop in mac VVQMLARLRKGYRPVFSSPPSY HQQILIHKGQEQPTKEGTEE I...K. .Q F.*THTQQDPAL..R..K.GD ......... .......... A.V ...
L.S.F ....
G.
..
P.
H
...
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P
DRG
AN.
.
FIG. 2. Comparative sequence analysis of env of SIVs and HIV-2. The predicted amino acid sequence of SIVstm was based on nucleotide sequences of PCR fragments amplified from EcoRI-digested SlVstm-infected human PBL DNA which were subsequently cloned into a vector DNA. Two cloned DNAs, ENV1.0 5 and ENV3 6, represented the SIVstm env sequence. This sequence was aligned with those of SIVmac25l (SIVmac), SIVsmmH4 (SIVsmm), and HIV-2BEN (HIV-2) (obtained from the Los Alamos HIV Sequence Database [1990]). Amino acid 1 corresponds to the amino acid at position 187 in the SIV/HIV-2 env consensus sequence. The designations, symbols, and alignment are as described in the legend to Fig. 1. The regions of greatest amino acid heterogeneity are boxed. Clusters 1 and 2 correspond to variable regions V4 and V5 (2), respectively.
VOL. 65, 1991
NOTES
7065
B 14.0
HUMAN
_
(1963) 8.5
STUMP-TAILED MACAOUE I
_
t
I
SIVSMMH4
SIVSTM-C( 1977) 1986 12.5
( 1974)
SIVSMMH4
1986
SIVMAC251
1986
1
12
10.
SIVMNE 1986
SOOTY MANGABEY
11
_58 57
17
SIVMAC142
SIVMNE
RHESUS MACAQUE PIG-TAILED MACAQUE
551
SIVMND MANDRILL FIG. 3. Phylogenetic relationships of lentiviruses of the SIV/HIV-2 group. (A) A minimum-length evolutionary tree was constructed from variation in the gag nucleotide sequence by using the PAUP computer program (4) with the global branch-swapping option MULPARB. The total number of sites examined was 1,386. The minimum length of the tree was 1,644, and the consistency index was 0.663. The tree was rooted at the midpoint of the greatest patristic distance. The indicated branch lengths are proportional to the minimum number of single-nucleotide substitutions necessary to generate the observed variations. The length of the vertical lines is for clarity only. (B) The number of years of divergence from a common ancestor, indicated above the branches, was determined as a fraction of the total number of years of divergence between any two isolates (calculated by dividing the sum of the branch lengths indicated in panel A by a fixation rate of 0.5% per year, the rate determined for HIV-1 gag [18]). The dates in parentheses represent estimated dates based upon the calculations. The dates not in parentheses are the dates of virus isolations. In the case of SIVstm, the calculated date is different from the date of virus isolation and coincides with the date when the tissue was stored in the freezer.
(This research was performed by Teresa A. Galvin in partial fulfillment of the requirements for a Ph.D. in genetics from George Washington University, Washington, D.C.) We are indebted to G. Myers for evolutionary tree analyses and thank M. A. Martin for critical review of the manuscript, N. Lerche for historical information regarding the CRPRC monkeys, J. Silver for helpful discussions, and A. Buckler-White for oligomer synthesIs.
1.
2. 3.
4.
5. 6.
7. 8.
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