Analysis of disease induction by simian immunodeficiency viruses
(SIV) in macaques was initially hampered by a lack of molecularly defined pathogenic strains. The first molecularly cloned SIV strains inoculated into macaques, SIVmacBK28 and SIVmacBK44 (hereafter designated BK28 and BK44, respectively), were cases in point, since
they failed to induce disease within 1 year postinoculation in any
inoculated animal. Here we report the natural history of infection with
BK28 and BK44 in inoculated rhesus macaques and efforts to increase the
pathogenicity of BK28 through genetic manipulation and in vivo passage.
BK44 infection resulted in no disease in four animals infected for more
than 7 years, whereas BK28 induced disease in less than half of animals
monitored for up to 7 years. Elongation of the BK28 transmembrane
protein (TM) coding sequence truncated by prior passage in human cells
marginally increased pathogenicity, with two of four animals dying in
the third year and one dying in the seventh year of infection.
Modification of the BK28 long terminal repeat to include four consensus
nuclear factor SP1 and two consensus NF-
B binding sites enhanced
early virus replication without augmenting pathogenicity. In contrast, in vivo passage of BK28 from the first animal to die from
immunodeficiency disease (1.5 years after infection) resulted in a
consistently pathogenic strain and a 50% survival time of about 1.3 years, thus corresponding to one of the most pathogenic SIV strains
identified to date. To determine whether the diverse viral quasispecies
that evolved during in vivo passage was required for pathogenicity or
whether a more virulent virus variant had evolved, we generated a
molecular clone composed of the 3' half of the viral genome derived
from the in vivo-passaged virus (H824) fused with the 5' half of the
BK28 genome. Kinetics of disease induction with this cloned virus
(BK28/H824) were similar to those with the in vivo-passaged virus, with
four of five animals surviving less than 1.7 years. Thus, evolution of
variants with enhanced pathogenicity can account for the increased
pathogenicity of this SIV strain. The genetic changes responsible for
this virulent transformation included at most 59 point mutations and 3 length-change mutations. The critical mutations were likely to have
been multiple and dispersed, including elongation of the TM and Nef
coding sequences; changes in RNA splice donor and acceptor sites, TATA
box sites, and Sp1 sites; multiple changes in the V2 region of SU,
including a consensus neutralization epitope; and five new N-linked
glycosylation sites in SU.
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INTRODUCTION |
The simian immunodeficiency viruses
(SIV) are a diverse collection of primate lentiviruses related to human
immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2, respectively),
with SIVmac (from macaques) and SIVsm (from sooty mangabeys)
together with HIV-2 comprising one of the five major primate lentivirus
lineages (24). The infection of macaques with SIVmac has
been widely used as a nonhuman model for investigating lentivirus
pathogenesis, transmission, tissue tropism, viral sequence variation,
antiviral therapy, and prophylaxis. Most molecularly cloned SIV have
been obtained following isolation in tissue cultures; several
SIVmac and SIVsm clones have been reported to express various
degrees of attenuated pathogenicity compared to the virus isolates from which they were derived (3, 17, 26, 31, 38), such as the
first SIV strain isolated, SIVmac251, which resulted in a 50%
1-year mortality rate in macaques (6, 7, 31). The molecular
bases for such observed attenuation of SIV clones remain largely
unknown.
After short-term evaluation of the initial molecularly cloned
SIVmac strains failed to produce disease (20, 38), we
sought to create pathogenic molecular clones of SIVmac to assist in
the development of this AIDS model system. In one approach, two
putative virulence determinants were investigated by the deliberate
introduction of specific mutations. Initially, the prematurely
truncated transmembrane protein (TM) gene of the clone SIVmacBK28
(29) was extended to encode full-length gp41
(21), similar to that found in HIV-1. We and others (5,
21, 28) showed that truncation and extension of the SIV TM
open reading frame (ORF) represented a reversible adaptation for
efficient replication in certain human cell lines and macaques,
respectively, but that extension of the TM ORF did not result in
acute disease induction (21, 28). Next, based upon the
observation that HIV-1 clones and the acutely lethal SIVsmmPBj14 contain duplicated NF-B sites and larger
numbers of near-consensus SP1-like recognition sites (11)
compared to SIVmacBK28 (29), we introduced reiterated
transcription factor binding sites in an effort to augment the
pathogenicity of SIVmacBK28. We now know that nef gene
expression is critical for disease induction (27) and that
changes in the nef gene that cause it to mimic the
nef gene found in SIVsmmPBj14 (11) greatly
enhance the virulence characteristics of the SIVmac239 clone
(13).
Experimental in vivo passage has often been found to increase the
virulence of laboratory-adapted virus isolates (16). Thus, our second approach for obtaining a pathogenic SIVmac clone was to
augment the virulence of an existing clone by in vivo passage and then
to isolate virus fragments bearing naturally selected mutations. The
second approach was successful, and by construction of a virus chimera
bearing a 3'-half genome from a macaque with immunodeficiency disease,
a pathogenic molecular clone was obtained.
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MATERIALS AND METHODS |
Viruses and cell lines.
For SIVmacBK28 and
SIVmacBK44, infectious stocks were prepared by transfection of the
molecular clones 
BK28 and BK44, derived from isolate
SIVmac251 (29), into HuT78 cells. For
SIVmacBK28-TM41, infectious stocks were prepared as previously
described (21) by transfection of the molecular clone
pBK28-TM41 into HuT78 cells. For SIVmacF965, bone marrow (obtained
at necropsy) from macaque F965 was cocultivated with human peripheral
blood mononuclear cells (PBMC) for 14 days, followed by passage of
cell-free virus on human PBMC for an additional 14 days. It should be
noted that this inoculum was prepared in 1988, prior to the publication
of studies demonstrating the selective pressures on the SIV TM coding sequence in vitro by human cells. Work by ourselves (21) and others (5, 28) revealed that rhesus PBMC provided a
less selective environment for SIVmac propagation than did human
cells, the latter of which were shown to select for genetic
alterations, including truncation of the TM gene. However, since the
inoculum was shown to be immediately pathogenic in vivo, we did not
need to recreate the inoculum by growth on macaque cells. For
SIVmacBK28/H824 and SIVmacBK28-2kB-4SP,
infectious stocks were prepared by liposome-mediated transfection of
plasmid DNA from pSIVmacBK28/H824 or linear concatameric pBK.1-5'half (see below) ligated with pBK28-3'-2kB-4SP into CEMx174 cells with Lipofectin reagent (GIBCO/BRL, Gaithersburg, Md.), according
to the manufacturer's instructions, and were passaged for 25 or 27 days, respectively. Cultures were monitored at 1- to 2-day
intervals for indications of virus production by visual evaluation of
syncytium formation and by a reverse transcriptase assay
(46). In all cases, virus-containing supernatants were passed through 0.22-µm-pore-size filters, mixed with equal volumes of
fetal calf serum, and then stored in liquid nitrogen. The amount of
inoculum used for each animal is shown in Fig.
1.
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FIG. 1.
Pathology in macaques infected with molecularly cloned
SIV strains. Age at inoculation is in years. An asterisk in the
survival column identifies macaques that were sacrificed while
clinically asymptomatic. Presentation of pathologic symptoms is
indicated by ×. Diagnoses were made either during disease or at
autopsy. Severities of lymphadenopathy and splenomegaly were graded by
distribution and size as mild (×), moderate (××), and severe
(×××). Thrombocytopenia was defined as platelet counts below
150,000/ml. Opportunistic infections (O.I.) associated with the
following pathogens were denoted as follows: CMV, cytomegalovirus; G.,
Giardia; B., Balantidium; T.,
Trichomonas; M.a., Mycobacterium
avium-intracellulare; B.b., Bordetella bronchiseptica;
C., Cryptosporidium; P.c., Pneumocystis carinii;
C.a., Candida albicans; S., Staphylococcus. Signs
of neurological involvement were identified as neuritis (N), peripheral
neuropathy (PN), spinal neuropathy (SN), meningitis (M), encephalitis
(E), and encephalomalacia (EM). Terminal levels of CD4+
cells/mm3 of blood were determined by flow cytometric
evaluation of samples stained with antibody FI-OKT4 in the clinical
laboratory and were expressed as both absolute cell numbers (Terminal
CD4 cell Numbers) and percentages of preinfection levels. N.D., not
determined.
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The HuT78 cell line was obtained from the American Type Culture
Collection (Rockville, Md.), and the CEMx174 cell line was obtained from the AIDS Research and Reference Reagent Program (Rockville, Md.). Both cell lines were maintained in RPMI medium supplemented with 10% heat-inactivated fetal calf serum, 100 U of
penicillin per ml, 100 µg of streptomycin per ml, and 2 mM L-glutamine.
Monkeys and in vivo inoculation experiments.
Indian rhesus
macaques (Macaca mulatta) used in these studies were housed
at the Tulane Regional Primate Research Center (TRPRC), Covington, La.,
and the Biomedical Primate Research Centre (BPRC), Rijswijk, The
Netherlands. Following intravenous inoculation (dose indicated in Fig.
1), animals were assayed for infection by nested PCR amplification of
PBMC DNA with primers specific for the viral long terminal repeat (LTR)
(34a), by measurement of SIV gp140-specific antibodies in
serum (42a), and by measurement of p27Gag levels
in serum with the SIV Core Antigen Assay (Coulter Immunology, Hialeah,
Fla.) (level of sensitivity, 0.03 ng/ml). Alternatively, animals were
assayed for infection on the basis of antigenic cross-reactivity with
HIV-1 by use of the Abbott HIV-1 Immunoassay (Abbott Laboratories, Abbott Park, Ill.) with incubation times modified for greater sensitivity (0.18 to 0.25 ng/ml) (9). Monkeys were observed daily by animal care staff, who noted any abnormal behavior. Full physical examinations were given by a veterinarian weekly for the
first 4 weeks and monthly thereafter. To assess clinical status, weight, lymph node and spleen sizes, body temperature, general physical
condition, and signs of opportunistic infections were monitored.
Complete blood counts and enumeration of lymphocyte subsets were
done periodically, and serum chemistries, fecal examinations, and
radiography were done when necessary for clinical diagnosis. Animals
which became moribund were humanely sacrificed.
Construction of viruses with additional NF-B and SP1 binding
sites in the 3' LTR.
Site-directed mutagenesis was performed as
previously described (19) to produce three synthetic LTRs,
one in which a second NF-B site was introduced, another in which the
original SP1-like motifs from clone SIVmacBK28 were replaced with
four consensus SP1 sites, and a third which contained both of these
changes (Fig. 2).
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FIG. 2.
Recombinant LTR enhancer constructs. (A) Sequence
alignments of SIVmacBK28 LTR U3 enhancer-promoter constructs and
the acutely pathogenic SIVsmmPBj4.41. Gaps introduced to maintain
alignment are indicated by dashes. Boxes enclose regions corresponding
to transcription factor binding site consensus sequences. The consensus
sequence (G/T)GGG(C/A)GG(G/A)(G/A)(C/T) was used to
delineate potential binding sites for SP1, and the consensus sequence
GGGACTTTCC was used to delineate potential binding sites for
NF-B. Bases which do not match the consensus sequence are indicated
above the sequence with an asterisk. (B) Schematic representation of
some molecularly cloned viruses used in this study. For the
construction of chimera pBK28-2kB-4SP, the enhancer element region
bounded by restriction sites NdeI and MscI was
replaced with a mutagenized fragment described in Materials and
Methods. For pBK28/H824, the 3'-half-genome fragments were PCR
amplified from thymus DNA from SIVmacF965-infected macaque H824 and
ligated into pUC19 to create 3'-half-genome clone pH824-3'. The 3'
BclI/EcoRI fragment was transferred into
5'-half-genome clone pBK.1-5' to create the full-length proviral
chimera.
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Mutagenesis was performed by adding 15 ng of plasmid pTG637 (a
derivative of pBK28 with only ~1.5 kb of flanking cellular DNA,
kindly provided by Marie-Paule Kieny) to 100 µl of a PCR reaction
cocktail containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.25 mM
MgCl2, 200 µM each deoxynucleoside triphosphate, 100 nM each relevant primer (Fig. 3), and 2.5 U
of AmpliTaq polymerase (Perkin Elmer/Roche Molecular Systems). Reaction
mixtures were overlaid with 100 µl of mineral oil and subjected to 15 cycles of amplification (94°C for 0.75 min, 60°C for 1 min,
and 72°C for 1 min). Mutant LTR fragments bearing two copies of the
NF-B binding sequence GGGACTTTCC were created with
primers PENF-kB2, PENF-kB7, PENF-kB1, and PENF-kB8. Similarly, LTR
fragments carrying four tandem repeats of the SP1 site GGGGAGGAGC
were created with PENF-kB9 and PENF-kB10 and with PENF-kB7 and
PENF-kB8.
The mutant LTR fragments were ligated into the EcoRI
and SalI sites of pBluescript KS+ (Stratagene, La Jolla,
Calif.), transformed into JM109 cells, and cultured at 37°C. Clones
bearing only the desired mutations were selected after DNA sequencing.
An LTR clone carrying two NF-B sites (pBK28-3'LTR-2kB) was used as
the template for an additional round of mutagenesis to combine the
NF-B and SP1 changes together in a single LTR by use of the SP1
mutagenic primers PENF-kB9 and PENF-kB10. The resulting product,
containing two NF-B sites and four SP1 sites, was cloned and
sequenced as described above.
Mutant LTRs containing two NF-B, four SP1, or both mutations were
transferred into a pBluescript KS+ clone carrying bases 9,171 to
10,249 of SIVmacBK28 to create mutant 3'-end clones. Each of
the mutant 3'-end clones was extended to span the entire 3' half of the
viral genome by insertion of the XbaI/BamHI
fragment (bases 4,076 to 9,171) from pBK28.
A clone containing the 5' half of the SIVmacBK28 viral genome
(pBK.1-5'half) was prepared by removal of the 2,350-bp ClaI fragment (bases 8,059 to 10,405) from pBK.1 (a full-length subclone of
pBK28 with only 150 bp of cellular flanking sequences, kindly provided
by G. Viglianti). The 5'-half-genome plasmid and each of the
3'-half-genome plasmids, pBK.1-3'half, pBK28-3'-2kB, pBK28-3'-4SP, and
pBK28-3'-2kB-4SP, were transformed into the dam host GM48. Infectious proviral DNA was assembled by digesting the 5'- and 3'-half-genome plasmids at the unique BclI site (base 5,113)
and then ligating them together to create linear concatamers as
previously described (11, 43). One month following
transfection, the integrity of the 3' LTR sequence was confirmed in
vivo by DNA sequencing (data not shown).
PCR amplification of 5-kb proviral half genomes.
Genomic DNA was prepared from cryopreserved thymus tissue from macaque
H824 by standard proteinase K digestion and phenol-chloroform extraction (49). To avoid preferential amplification of
proviruses bearing large internal deletions, the genomic target DNA was
diluted prior to amplification (14). One hundred nanograms
of thymus DNA was used as a template in 100-µl PCR reaction mixtures
containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM
MgCl2, 200 µM each deoxynucleoside triphosphate, 1%
dimethyl sulfoxide, 100 nM (each) primers SD3 and SD2 (Fig. 1), and 2.5 U of AmpliTaq polymerase. Reaction mixtures were overlaid with 100 µl
of mineral oil and subjected to 35 cycles of amplification (94°C for
0.75 min, 55°C for 1 min, and 72°C for 4 min) followed by 72°C
for 10 min (first round) in a Thermal Cycler (Perkin-Elmer, Foster
City, Calif.). Two microliters from each reaction mixture was
transferred to fresh reaction tubes containing PCR reaction mixtures
plus 100 nM (each) primers SD3 and SD9 (Fig. 3) and then reamplified
for an additional 35 cycles under the same conditions (second round). As controls for contamination, reactions containing normal human DNA
extracted in parallel with the H824 sample and reagents only were
performed simultaneously. Positive reactions were identified by agarose
gel electrophoresis with ethidium bromide staining.
Construction of chimeric provirus pBK28/H824.
3'-half-genome
PCR products, amplified as described above from postmortem thymus DNA
from macaque H824, were digested with EcoRI (at sites
encoded by the PCR primers SD9 and SD12), ligated into the
EcoRI site of pUC19, and then electroporated into 40 µl of
J5 cells as previously described (1) (kindly provided by V. Hirsch, Georgetown University). Electroporation was performed at 400 
, 2.5 kV, and 25 µF in 0.2-cm electroporation cuvettes with a Gene
Pulser and a Pulse Controller (Bio-Rad, Hercules, Calif.).
Electroporated cells were incubated at 37°C for 1 h with Luria-Bertani medium and then spread onto Luria-Bertani agar plates containing 100 µg of carbenicillin per ml. Bacterial incubation on solid and in liquid media was performed at room temperature rather
than at 37°C to enhance the stability of plasmids containing large
lentivirus inserts (30; data not shown). The
3'-half-genome plasmid pH824-3' was electroporated into the
dam bacterial strain GM48.
The 5'-half-genome subclone pBK.1-5' was derived from pBK.1 by
replacement of the 3' 5.3-kb BclI/EcoRI fragment
(bases 5,113 to 10,404) with a BclI/EcoRI adapter
made by annealing two partially complementary oligonucleotides, PE
BclI-Eco-L1, and PE BclI-Eco-L2 (Fig. 3), which combined to create a
16-bp double-stranded DNA fragment with 5' overhangs that are
complementary to the BclI and EcoRI sites,
respectively. Full-length chimeric provirus pBK28/H234-3' was
constructed by ligation of the 3'-half-genome fragments excised from
pH824-3' plasmids 1 to 5 (SIVmacBK28 nucleotide positions 5,113 to
10,189) into the BclI and EcoRI sites of plasmid
pBK.1-5'.
DNA sequencing.
The complete nucleotide sequence of clone
pBK28 was determined by deletion clone generation in phage M13 with the
Bal 31 method and 35S-dideoxynucleotide
sequencing. The sequence of the H824 3'-half genome was determined by a
shotgun strategy of subcloning into an M13 vector with subsequent
sequencing by use of Taq polymerase and fluorescent
dideoxynucleotides, and the products were resolved with an Applied
Biosystems 370A DNA sequencer. The nucleotide sequences of shorter
segments of env were determined as part of another study
(14a).
Nucleotide sequence accession numbers.
The pBK28 sequence
has been deposited in GenBank under accession numbers including
M15897. The H824 3'-half-genome sequence has been deposited in GenBank
under accession no. U86638.
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RESULTS |
Infection of macaques with molecularly cloned SIVmacBK28
and SIVmacBK44: first in vivo passage.
Over the course
of several studies to evaluate the natural history of infection and
virus stock titration, a total of 24 rhesus macaques at the TRPRC and
the BPRC were infected with the molecularly cloned virus
SIVmacBK28. Fourteen animals were sacrificed for tissue collection
in the absence of overt manifestations of AIDS-like disease between 15 and 896 days postinfection (p.i.). Among the remaining 10 macaques, the
median survival exceeded 7 years (range, 520 to >3,200 days) (Fig.
4A) and included the deaths of 5 macaques with AIDS-like symptoms after 520 to 1,565 days (4 of which are described in Fig. 1). Four additional macaques at the TRPRC were infected with SIVmacBK44, a clone derived from the same culture as
SIVmacBK28 (29). None of these macaques died during the
7- year follow-up (Fig. 4A), and none developed p27Gag
antigenemia within 2 weeks p.i. (Fig. 1 and Table
1).
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FIG. 4.
Macaque survival after SIV infection. Graphs are
Kaplan-Meier depictions of survival of SIV-infected macaques over
time.
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Infection with SIVmacBK28 was generally characterized by
asymptomatic infection and long-term survival with, on average,
increasing levels of CD4+ cells (Fig.
5). Four of 16 animals tested developed
viral p27Gag antigenemia 2 weeks p.i. (Table 1). The most
common clinical findings in diseased macaques were mild lymphadenopathy
and splenomegaly, with cachexia, enteritis, and thrombocytopenia being
less frequently observed (Fig. 1). Opportunistic pathogens were
detected in three macaques (Fig. 1). Hence, the molecular clones
SIVmacBK28 and SIVmacBK44, derived following adaptation of the
virus to growth in the human T-cell line HuT78, appeared to be less
pathogenic than the SIVmac251 isolate from which they were derived.
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FIG. 5.
Median CD4+ cell levels in rhesus monkeys
infected with four different SIV clones. Absolute numbers of
circulating CD4+ cells were measured in rhesus
monkeys infected with SIVmacBK28 ( ), SIVmacF965 ( ),
SIVmacBK28-2kB-4SP ( ), or SIVmacBK28/H824 ( )
and expressed relative to preinfection levels. Median values were
calculated from all of the individual animal measurements (3 to 20 values for each point). Correlation coefficients (standard errors of
the mean) were 0.30 (0.0033), 0.27 (0.001), 0.46 (0.0019), and 0.62 (0.0013) for animals infected with SIVmacBK28,
SIVmacBK28-2kB-4SP, SIVmacBK28/H824, and SIVmac965,
respectively.
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Genetic manipulation of SIVmacBK28 in attempts to enhance
pathogenicity. (i) Removal of the stop codon in the TM.
The
portion of the env gene ORF encoding the TM is truncated as
a result of adaptation to growth in some human T-cell lines (5,
21, 28). This ORF was extended by removal of the premature stop
codon (21), and four macaques at the TRPRC were infected with this virus (SIVmacBK28-TM41). Median survival was reduced to
2.5 years p.i. (Fig. 4B); hence, a small increase in pathogenicity in
vivo could have resulted from this change.
(ii) Effect of transcription factor site reiteration on
pathogenicity.
The acutely lethal SIV pathogen, SIVsmmPBj14,
was previously found to contain a second NF-B binding site not found
in other SIV strains (11). A further comparison to the
SIVmacBK28 LTR sequence also revealed that it had fewer and less
consensus-like nuclear factor SP1 binding sites (11, 29)
(Fig. 2). Since similar changes had been known or suspected to have an
impact on retrovirus pathogenicity (8, 10, 52), we sought to
investigate the effect of NF-B and SP1 binding site reiteration on
the pathogenicity of SIVmacBK28. A series of mutant viruses
containing different combinations of these sites in the 3' LTR were
generated. With site-directed mutagenesis, three variant LTRs were
constructed, the first with two NF-B sites and wild-type
SIVmacBK28 SP1 sites, the second with four consensus SP1 sites and
a single wild-type NF-B site, and the third with both two NF-B
sites and four consensus SP1 sites; the placement of each site was
chosen to match that found in the SIVsmmPBj14 sequence (Fig. 2).
The wild-type and mutant LTRs were placed into the context of the
full-length viral genome and transfected into cells to produce stocks
of infectious virus. All four constructs displayed similar in vitro
phenotypes characterized by syncytium induction and replication to high
reverse transcriptase levels in HuT78 cells (data not shown).
Mutant virus derived from the construct containing the maximum
number of NF-B and SP1 sites (SIVmacBK28-2kB-4SP) was
used to infect five rhesus macaques at the TRPRC to evaluate its
pathogenicity in vivo. Four of the five were antigenemic on day 13 p.i. (Table 1), although none experienced acute onset of clinical
disease. All were PCR positive for proviral DNA from PBMC at 285 days
(data not shown). Two animals (K128 and K163) died with AIDS-like
symptoms after 819 and 923 days, respectively (Fig. 1 and 4B).
Throughout 125 weeks of hematological follow-up, levels of circulating
CD4+ cells (and CD4+CD29+
lymphocytes; data not shown) in SIVmacBK28-2kB-4SP-infected
macaques remained stable on average although depressed relative to
levels in macaques infected with wild-type SIVmacBK28
(P < 0.05) (Fig. 5). However, the two rhesus macaques
that died (K128 and K163) experienced greater than 80% declines in
CD4+ lymphocyte levels, while CD4+ cell
populations in the remaining macaques fluctuated near or above
preinfection levels (data not shown). The induction of acute antigenemia in 80% of the animals infected with SIVmacBK28-2kB-4SP contrasted with the results for those infected with the parental virus
SIVmacBK28 (25% of the animals; P < 0.025) (Table
1). Thus, replacement of the wild-type enhancer with a sequence
containing two NF-B and four consensus SP1 binding sites appeared to
have enhanced early virus replication without substantially augmenting pathogenicity.
Second in vivo passage of SIVmacBK28: infection
with SIVmacF965.
The deaths of three macaques in less
than 2 years after infection with SIVmacBK28 contrasted with the
long-term asymptomatic survival more typical of infection with this
virus and suggested that virulent viruses or virus mixtures may have
developed within these relatively short-lived animals. To test this
hypothesis, virus was isolated from the bone marrow of the macaque with
the shortest survival (520 days p.i., animal F965), expanded in vitro through two 14-day passages in human PBMC, and inoculated into a second
group of macaques.
Twenty macaques at the TRPRC were inoculated with isolate
SIVmacF965. In contrast to the results for animals inoculated with the parental molecularly cloned virus, death with AIDS-like symptoms occurred in 95% of infected macaques within 100 to 841 days
(median, 457 days), with 85% (17 of 20) dying by 2 years p.i. and only one surviving more than 2.3 years (Fig. 1 and 4C). Thus, overall median
survival was reduced about threefold from approximately 1,500 days with
SIVmacBK28 infection to 470 days (one-tailed t test, assuming unequal variance; P < 0.005). As also
found previously (40), survival was independent of
infectious dose over a 1,000-fold range (Fig. 1). Furthermore, all
SIVmacF965-infected macaques were positive for viral
p27Gag antigenemia during acute infection (Table 1). Among
eight animals examined in detail, antigenemia persisted throughout
infection in the most rapidly declining animals, whereas clearance of
antigen or clearance followed by a return of antigen was seen in the
remaining macaques examined (data not shown). Production of anti-SIV
antibodies was undetectable by Western blotting throughout infection in
the macaques with the shortest survival, while broad antibody responses against several SIV proteins (p17Gag, p27Gag,
gp35/41Env, and gp120Env) were detected in the
macaques with longer survival (data not shown).
The majority of SIVmacF965-infected macaques with clinical disease
displayed marked declines in CD4+ cell levels (and
CD4+CD29+ cell levels; data not shown) relative
to animals infected with SIVmacBK28 and SIVmacBK28-2kB-4SP
(P < 0.05) (Fig. 5). Common symptoms were moderate to
severe lymphadenopathy, splenomegaly, cachexia, enteritis, renal and
hepatic disease, arthritis, and rashes (Fig. 1). Thrombocytopenia was
present in 9 of 15 macaques that survived more than 300 days but was
not detected in those with shorter survival. Opportunistic pathogens
were detected in eight macaques, and lymphomas were found in three
(Fig. 1).
Infection with chimeric virus BK28/H824.
We next evaluated the
hypothesis that the enhanced pathogenicity of the SIVmacF965 strain
could be embodied within individual virus variants rather than
requiring a pool of equally virulent but antigenically diverse variants
for rapid disease induction. Chimeric proviruses were generated by
combining the 5' half of SIVmacBK28 (nucleotide positions 1 to
5,113) with four distinct 3'-half genomes (nucleotide positions 5,113 to 10,189) obtained by direct PCR amplification of thymus DNA from
SIVmacF965-infected macaque H824 (Fig. 2). This macaque was
chronologically the first to die following infection with
SIVmacF965, at 173 days p.i. Each chimera was transfected into
CEMx174 cells, but only one (hereafter referred to as BK28/H824) gave
rise to infectious virus, as measured by reverse transcriptase
activity, syncytium induction, and cell-free infectivity (data not
shown). BK28/H824 displayed a phenotype in cultures distinct from that
of SIVmacBK28. While both replicated to high reverse transcriptase
levels and produced large multinucleated syncytia in CEMx174 cells,
BK28/H824 did not replicate in HuT78 cells, even after deliberate
truncation of the TM from 41 to 32 kDa (data not shown).
Five rhesus macaques at the TRPRC were inoculated with the molecularly
cloned chimeric virus BK28/H824. All five became antigenemic, with measurable serum p27Gag levels on day 13 p.i. but
at a mean level lower than that found in SIVmacF965-infected
animals (Table 1). Death with AIDS-like symptoms occurred in four of
these infected macaques within 393 to 635 days (median, 462 days), with
only animal L643 surviving more than 2 years (Fig. 1 and 4C). Symptoms
were similar to those detected following infection with the ancestral
SIVmacF965 isolate (Fig. 1).
Coincident with acute antigenemia were marked declines in circulating
CD4+ cell levels in all infected animals beginning at 2 weeks p.i. (Fig. 5) as well as in CD4+CD29+
cell levels (data not shown). During the first 2 years of hematological follow-up, four of the five macaques experienced large declines (greater than 40 to 80%) in circulating CD4+ and
CD4+CD29+ cell levels (Fig. 5; see Fig. 1 for
data from individual animals). On average, CD4+ and
CD4+CD29+ cell levels in BK28/H824-infected
macaques were depressed relative to levels observed following
SIVmacBK28 and SIVmacBK28-2kB-4SP infections (P < 0.05), although less so than following infection with SIVmacF965
(Fig. 5).
Nucleotide changes in the H824 3'-half genome associated with
disease induction.
Fifty-nine point mutations over 5,111 bases
(1.15% divergence), 12- and 1-base deletions, and a 3-base insertion
distinguished the 3'-half genomes of H824 and BK28 (Fig.
6). A summary of these changes is shown
in Table 2. Several changes that might
conceivably contribute to the increased virulence of the BK28/H824
chimera were found. Mutations were noted in the consensus splice donor for tat and rev and in the acceptor for
tat, rev, and nef, the latter of which
also extended the TM coding sequence to encode a putative gp41 versus
the gp32 encoded by BK28.
View larger version (65K):
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|
FIG. 6.
Deduced protein and LTR sequences from clone H824
compared to those of parental clone BK28. Sequences that differ from
the BK28 sequence are indicated for each sequence element, whereas
identical sequences are indicated by dots and gaps are indicated by
dashes.  Pol and LTR indicate that the N-terminal sequence of Pol
and the 3' end of the LTR were not replaced in the BK28/H824 chimera.
Predicted N-glycosylation sites that differ between the two sequences
are underlined. A summary of the changes is shown in Tables 2 and 3.
|
|
As a result of a single base deletion, BK28 encoded a truncated Nef
protein with a nonconsensus string of 15 amino acids following the
frameshift at the C-terminal end. This mutation was exquisitely repaired in vivo by the insertion of the consensus (T) nucleotide in
the H824 sequence. In the LTR, the TATA box consensus sequence was
changed from TATAA to CATAA, and two mutations were noted in Sp1 sites,
an A
G mutation in position 10 of SP1 0, a change that similarly
agrees with the consensus, and a deletion of a G between Sp1 III and
Sp1 III, which reduces the spacing between these sites (Fig. 2A).
Env glycosylation site changes.
Mutations resulting in the
loss of one and the gain of five new sites for potential N-linked
glycosylation occurred within the region from V1 to V5 of the H824
gp120 coding sequence. Given the involvement of N-linked glycosylation
in virus replication and host interactions (2, 15, 18),
conservation of these sites was evaluated in the other characterized
SIV clones as well as BK28-infected macaques to investigate a possible
association with SIV pathogenicity. We used DNA sequence analysis
(14a) and allele-specific amplification (39)
(Table 3). The site at position 114 was
quantitatively lost by 267 days following infection in animal F965
(Table 3), as a result of an SN change that simultaneously created
the new site at position 116. The same mutation occurred during viral
growth within two of the three other animals evaluated (G015 and I049
but not 2BS). Animal 2BS was the only other animal of this group to
succumb to an AIDS-like illness; hence, a shift from position 114 to
position 116 was not closely linked to disease induction in this study.
The N-linked glycosylation site at position 173 was found in all of the
other SIV strains evaluated and in 100% of the viral genomes evaluated
in animals F965 and 2BS, at 267 and 686 days after infection,
respectively, and in animal I049, when sacrificed while asymptomatic
896 days p.i. This site was not found in animal G015 at 724 days p.i.;
this animal was later sacrificed when asymptomatic more than 8 years
after infection. A significant correlation was found between the
presence of this site and the proviral DNA load in the PBMC of infected
macaques. The site was found in five of six macaques with high virus
loads (50 to 240 proviruses/105 cells, including 2BS, I049,
and F965) but in only one of seven macaques with low provirus loads
(0.6 to 10 proviruses/105 cells, including G015)
(chi-square test, P < 0.025).
Similar patterns of acquisition were found at N-linked glycosylation
sites at positions 417 (100% conversion in 2BS, I049, and F965 but
37% in G015) and 481 (100% in 2BS, I049, and F965 and 88% in G015),
although the larger set of animals for which provirus load was known
was not evaluated for the presence of these sites. The site at 417 was
also acquired by virus within a subset of macaques infected with the
pathogenic molecular clone SIVmac239, the parental virus of which
also lacked the glycosylation sites at positions 256 and 417 (Table 3)
(4). The pattern of all six sites appearing in
SIVmacF965 was generally maintained through in vivo passage of this
strain (Table 3).
| |
DISCUSSION |
The initial objective of this study was to obtain a
pathogenic molecular clone of SIVmac to be used for the
investigation of SIV pathogenesis. Initial animal inoculations with
molecular clones SIVmacBK28 and SIVmacBK44 resulted in
long-term asymptomatic survival and no disease at all
induced by SIVmacBK44, even after follow-up for up to 7 years. SIVmacBK44 thus appears to embody a truly minimally
pathogenic phenotype. Three approaches were used to enhance the
pathogenicity of the slightly more pathogenic SIVmacBK28: (i)
extension of the env TM coding sequence truncated by passage
in a human T-cell line (HuT78), (ii) deliberate introduction of
specific mutations designed to augment the LTR enhancer, and (iii) in
vivo passage followed by cloning and testing of virus fragments bearing
naturally selected mutations.
Extension of the TM may have resulted in some enhancement of
pathogenicity. The gene reverts rapidly to an extended form in vivo
(21, 28), indicating that the truncated form of the protein results in attenuated virus replication in vivo. Thus, the slightly enhanced pathogenicity of the virus may have been due to a more fulminant replication early in infection, although the latter was not
reflected in the detectable production of p27Gag, since
none of the animals infected with SIVmacBK28-TM41 became antigenemic.
The introduction of a second NF-B binding site and replacement of
the three sequences resembling SP1 binding sites with four consensus
SP1 binding sites in the SIVmacBK28 LTR enhancer simulated the LTR
structure of the acute pathogen SIVsmmPBj14 (11). This change increased virus replication early after infection, reflected in
the detection of antigenemia, but only marginally enhanced viral
pathogenicity in vivo. These results are in agreement with those of
other studies indicating that the acute pathogenicity of
SIVsmmPBj14 was not due to changes in the LTR alone (41) but rather was due primarily to point mutation changes within the
nef gene (13).
Passage of SIVmacBK28 in vivo resulted in the development of a
consistent SIV pathogen derived from a human PBMC coculture of bone
marrow cells from the first animal to die from this inoculum, 17 months
p.i. (isolate SIVmacF965). Passage of the SIVmacF965 virus
resulted in 85% mortality within 2 years in a second group of
macaques. Unclear at this point was whether viral genetic and antigenic
diversity was required for disease induction, as would be predicted by
the "Lilliputian" hypothesis or "antigenic threshold" theory
for AIDS pathogenesis (35, 42), or whether outgrowth of
"virulent variants," as has been found for
immunodeficiency-inducing feline leukemia virus variants (23, 35,
36, 44), was responsible for the increased pathogenicity of the
strain. The latter hypothesis was supported by the next finding that,
like the uncloned progenitor virus SIVmacF965, a BK28/H824 virus
chimera in which the 3'-half genome was derived from an animal infected
with the SIVmacF965 strain resulted in the death of 80% of
infected macaques with AIDS-like symptoms within 2 years. Thus, the
BK28/H824 chimera embodied the essential pathogenic potential of the
uncloned SIVmacF965 isolate and localized the major pathogenic
determinants to the 3' half of the viral genome.
The appearance of putative N-linked glycosylation sites in SIV Env
suggests that they may contribute to the pathogenic phenotype of H824.
However, the prevalence of the N-linked glycosylation sites at
positions 116, 173, 256, 417, and 481 in other SIV strains, including
the minimally pathogenic strain SIVmac1A11 (34), and the
maintenance of these sites upon passage of the SIVmacF965 strain in
vivo are also consistent with the hypothesis that they may be natural
features of SIV Env. It is possible that SIVmacBK28 lost some of
these N-linked glycosylation sites during prolonged passage in a
transformed human cell line prior to its molecular cloning
(29) and that those sites were later recovered as the virus
readapted to replication in vivo. Thus, such changes may be necessary
for increased virus replication but not sufficient for enhanced
pathogenicity.
One of the changes that occurred during the evolution of the H824
pathogen was also deliberately introduced in parallel experiments designed to increase the pathogenicity of BK28 (i.e., extension of the
TM coding sequence in SIVmacBK28-TM41). Some changes also occurred in the enhancer region in vivo, distinct from but within a
region modified in a second deliberate construct,
SIVmacBK28-2kB-4SP. Whereas neither deliberately introduced change
was sufficient to confer the full pathogenic phenotype, at least the TM
extension was likely to have contributed, since viruses with only this
change were slightly more pathogenic than parental virus BK28. It is likely, therefore, that H824 corresponds to a relatively highly evolved
pathogen, with multiple mutations contributing to its more pathogenic
phenotype. Similarly, when the roles of individual mutations in the
phenotype of the feline leukemia virus-feline AIDS pathogen were
dissected, a series of both essential and virulence-enhancing mutations
were identified (12).
As in the current study, in vivo passage of primate lentiviruses,
especially serial passage, has been found to yield virus isolates with
properties distinct from those of the parental viruses. The acutely
pathogenic SIVsmmPBj14 isolate was derived following a single in
vivo passage of the minimally pathogenic SIVsmm9 (17), and molecular clones of that isolate have been shown to recapitulate the rapidly fatal phenotype (11). Increased neurovirulence
was observed following serial transfer of SIVmac239 from bone
marrow to brain in macaques (51), and serial intravenous
passage of microglia-associated virus following infection of rhesus
macaques with uncloned SIVmac251 yielded a virus stock with
neuroinvasive and neurovirulent properties (53). Increased
pathogenicity was also observed during serial passage of virus from
blood to blood following infection of a rhesus macaque with the
minimally pathogenic clone SHIV-89.6 (carrying env from a
T-cell and macrophage-tropic HIV-1 clone) (47). Similarly,
increased virulence was detected following each serial transfer of the
minimally pathogenic clone SHIV-HXBc2 from bone marrow to bone marrow
in pigtailed macaques (25). However, while in vivo passage
has been used on several occasions to obtain more highly pathogenic SIV
or simian-human immunodeficiency virus (SHIV) isolates, in few cases
have pathogenic molecular clones resulted.
Studies of HIV-1-infected individuals reveal that viruses that
appear to be more virulent in vitro (by virtue of their broadened host
range and cytopathogenicity associated with fast replication and
syncytium formation) are found in about half of individuals progressing
to AIDS but not in long-term asymptomatic individuals (50).
The fact that syncytium-inducing variants are not found in all
individuals progressing to AIDS raises the concern that they arise only
following the failure of the immune system rather than being a cause of
the failure. Furthermore, the relentless, massive turnover of virus
that occurs in HIV-1-infected individuals (22, 45, 54)
emphasizes the fact that minor changes in the growth properties of the
virus could have a major impact on the delicate balance of virus and
the available target cell reservoir (48); indeed, the
diversity of the virus population present within an infected individual
can be enormous. However, direct analysis of viral quasispecies in
HIV-1-infected individuals reveals a correlation between outgrowth of a
relatively uniform virus population in quickly progressing individuals
and more rapid and greater diversification of the virus population in
slowly progressing or nonprogressing individuals (32, 33,
55). The data from the present study provide a precedent that the
formation of a diversified lentivirus population, while one of the more
insidious properties thought to be crucial to escape from the immune
system and fulminant persistence in the host, is not a prerequisite for the development of AIDS.
This work was supported by grants from the U.S. Public Health
Service.
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Abstract
Introduction
