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Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4701-4707
Selective Ablation of Human T-Cell Lymphotropic Virus Type 1 p12I Reduces Viral Infectivity In Vivo
By
Nathaniel D. Collins,
Garret C. Newbound,
Björn Albrecht,
Jennifer L. Beard,
Lee Ratner, and
Michael D. Lairmore
From the Center for Retrovirus Research and the Department of
Veterinary Biosciences, The Ohio State University, Columbus, OH; the
Departments of Medicine, Pathology, and Molecular Microbiology,
Washington University School of Medicine, St Louis, MO; and the
Comprehensive Cancer Center, The Arthur James Cancer Hospital and
Research Institute, The Ohio State University, Columbus, OH.
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ABSTRACT |
Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic
agent of adult T-cell leukemia and HTLV-1-associated myelopathy. Novel,
yet conserved RNA transcripts encoded from open reading frames (ORFs) I
and II of the viral pX region are expressed both in vitro and in
infected individuals. The ORF I mRNA encodes the protein
p12I, which has been shown to localize to cellular
endomembranes, cooperate with bovine papillomavirus E5 in
transformation, as well as bind to the IL-2 receptor  and  chains
and the H+ vacuolar ATPase. It is unknown what role
p12I plays in the viral life cycle. Using an infectious
molecular clone of HTLV-1 (ACH) and a derivative clone,
ACH.p12I, which fails to produce the p12I
message, we investigated the importance of p12I in infected
primary cells and in a rabbit model of the infection. ACH.p12I was infectious in vitro as shown by viral passage
in culture and no qualitative or quantitative differences were noted
between ACH and ACH.p12I in posttransfection viral antigen
production. However, in contrast to ACH, ACH.p12I failed to
establish persistent infection in vivo as indicated by reduced
anti-HTLV-1 antibody responses, failure to demonstrate viral p19
antigen production in peripheral blood mononuclear cell (PBMC)
cultures, and only transient detection of provirus by polymerase chain
reaction in PBMC from ACH.p12I-inoculated rabbits. These
results are the first to show the essential role of HTLV-1
p12I in the establishment of persistent viral infection in
vivo and suggest potential new targets in antiviral strategies to
prevent HTLV-1 infection.
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INTRODUCTION |
HUMAN T-CELL lymphotropic virus
type 1 (HTLV-1) has been identified as the causative agent of adult
T-cell leukemia and appears to initiate a variety of immune-mediated
disorders including the chronic degenerative disease, HTLV-1-associated
myelopathy/tropical spastic paraparesis.1,2 This complex
retrovirus contains, in addition to the typical retroviral structural
genes gag, pol, and env, several regulatory genes. These regulatory
genes are derived from four open reading frames (ORFs) in the pX region located between env and the 3 LTR. ORFs IV and III encode the well-characterized Tax and Rex proteins, respectively.3
Tax, a 40-kD transactivating protein has been implicated in viral
pathogenesis, in part, by dysregulation of a number of important
regulatory cellular proteins including CREB/ATF, NF- B, SRF, and
p16INK.4 Rex, a 27-kD phosphoprotein, is
critical in RNA processing and the production of viral structural
proteins.5 Less clear is knowledge of host factors and
other viral genes important in establishment and pathology of
persistent HTLV-1 infections.
It is uncertain what role, if any, pX ORFs I and II play in viral
replication or pathogenesis. Several groups have identified conserved
ORF I and II mRNA species by reverse transcriptase-polymerase chain
reaction (RT-PCR) and RNAase protection assay in HTLV-1-infected cells
and individuals, but have been unable to show expression of protein
encoded by these messages.6-9 However, expression of
protein, including p12I, p13II, and
p30II, has been achieved upon transfection of ORF I- and
II-containing plasmids into eukaryotic cells.7,8,10 The
p12I protein has been shown to be highly hydrophobic and
localized to cellular endomembranes.10 This 99-amino acid
protein contains four SH3 binding motifs, cooperates with bovine
papillomavirus E5 in transformation, associates with the H+
vacuolar adenosine triphosphatase (ATPase), and may
decrease expression of the interleukin-2 receptor (IL-2R) and
c chains.11-13
Despite the evidence for p12I functionality, the role of
this protein in the viral life cycle has not been established, although recent evidence suggests that the p12I message is not
required for viral infectivity, replication, or transformation in vitro
(Michael Robek et al, personal communication, October,
1997). However, large deletions of regions in bovine leukemia virus and HTLV-2 analogous to HTLV-1 ORF I and II resulted in
lower viral loads in vivo, although no deleterious effect on virus
production or transforming capacity was detected in
vitro.14-16 We have recently shown a full-length clone of
HTLV-1, designated ACH, to be infectious in vitro and in vivo in a
rabbit model.17 By using ACH and a derivative clone in
which the splice acceptor site for the third exon of the ORF I message
is destroyed, we examined the specific role of p12I in
viral replication and infectivity in vitro and in vivo. Upon transfection into human peripheral blood mononuclear cells (PBMC), we
found no differences between ACH and ACH.p12I in virus
production despite the absence of the p12I message in
ACH.p12I-transfectants. Upon inoculation into rabbits, ACH-
and ACH.p12I-transfectants both induced anti-HTLV-1
antibody responses, but those of ACH.p12I-inoculated
rabbits were weaker. Furthermore, we were able to consistently isolate
virus only from ACH-inoculated animals, indicating that HTLV-1
p12I is important for viral infectivity in vivo. These data
provide the first evidence of a functional role of p12I in
establishing persistent HTLV-1 infections and suggest a novel target
for antiviral therapies.
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MATERIALS AND METHODS |
Plasmids and transfection of PBMC.
Normal uninfected human PBMC were obtained by leukophoresis as
previously described.18 PBMC were maintained in RPMI 1640 supplemented with 15% fetal bovine serum, 0.3 mg/mL L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 U/mL rIL-2 (complete
media). Before transfection, PBMC were stimulated with 2 µg/mL
phytohemagglutinin-P (PHA-P) and cultured for 4 days. Cells were then
transiently transfected by electroporation as previously
described18 using 10 µg of either the HTLV-1 molecular clone ACH,19 ACH.p12I, a construct in which a
Pst I site corresponding to the splice acceptor for the coding
exon of the p12I message has been deleted
(Fig 1), or the pKS vector (Stratagene, La Jolla, CA) only. Transfected cells were then
restimulated with 2 µg/mL PHA-P, seeded in 24-well plates, and
maintained in complete RPMI, with media changes and expansion of
cultures to 6-well plates as needed.
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| Fig 1.
Schematic representation of spliced transcripts of
HTLV-1. ORFs are represented by open boxes. Location of  PstI
introduced in ACH.p12I is indicated and corresponds to the
splice acceptor for exon 3 of the ORF I message. Primers used in PCR
and RT-PCR are indicated above the full-length provirus. Numbers
correspond to nucleotide positions with respect to the ACH
clone.19
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Detection of viral p19 matrix antigen.
As a comparison of virus production between ACH- and
ACH.p12I-transfectants, culture supernatants were sampled
weekly and assayed for p19 matrix antigen using a commercially
available enzyme-linked immunosorbent assay (ELISA; Cellular Products,
Buffalo, NY), with a detection sensitivity of 25 pg/mL p19
protein. Resultant absorbance values were compared with a standard
curve generated in the same assay. Values were statistically compared
by one-way analysis of variance (ANOVA).
For detection of HTLV-1 p19 antigen ex vivo, rabbit PBMC were isolated
from whole blood by density gradient (Lympholyte Rabbit; Cedarlane,
Hornsby, Ontario, Canada), stimulated with 3 µg/mL Concanavalin A (Con A; Sigma, St Louis, MO) and cultured in complete RPMI. Culture supernatants were collected at day 7 and assayed for p19
antigen as described above.
Syncytia assay.
As an indirect measure of transfectant viral envelope production, a
syncytia assay was performed as previously described.20,21 Briefly, human PBMC transfectants were washed, resuspended at 5 × 105/mL, and duplicate aliquots placed into individual wells
of a 96-well plate containing confluent human osteosarcoma (HOS) cells. After a 24-hour incubation, plates were washed, Wright's stained, and
examined for syncytia containing at least four nuclei per cell. Data
were expressed as the number of syncytia per well and values were
statistically compared by ANOVA.
Infectivity assay.
Infectious virus production by transfectants was assayed by coculturing
1 × 106 naïve rabbit PBMC with 1 × 105 gamma-irradiated (5,000 rad) transfectant
cells in individual wells of a 24-well plate. Irradiated transfectants
were also cultured alone to control for cessation of viral antigen
production by irradiated cells. After maintenance in complete RPMI for
2 weeks, cells were washed to ensure measurement of de novo antigen
production and cultured in fresh 24-well plates for 7 days, with
aliquots of culture supernatant obtained at days 1 and 7 and assayed
for p19 antigen as described above.
Detection of proviral sequences and viral mRNA.
For detection of provirus in transfectants or rabbit PBMC, genomic DNA
was obtained by affinity column (QIAamp; Qiagen, Santa Clarita,
CA) and examined for the presence of HTLV-1 nucleotide sequences by PCR. Five hundred nanograms of DNA was amplified using a
primer pair specific for the HTLV-1 pX ORF I region (6555, 5-AGGACCATGCATCCTCCGTCAG-3; 7492, 5-AGCCGATAACGCGTCCATCGAT-3), which yielded a 938-bp
product that included the Pst I deletion site of
ACH.p12I at nucleotide 6732. As a positive control and to
provide for semiquantitative comparison of HTLV-1 products,
simultaneous amplification was performed with a primer pair specific
for -actin,15 which yielded a 415-bp
product from rabbit DNA. After an initial 10-minute incubation at
95°C to activate the Taq polymerase (AmpliTaq Gold; Perkin-Elmer, Foster City, CA), 35 cycles of PCR were
performed with the following cycle parameters: denaturation at 94°C
for 1 minute, annealing at 55°C for 2 minutes, and extension at
72°C for 2 minutes, followed by a final extension at 72°C for 5 minutes. The amplified products were separated in a 1.5% agarose gel
and stained with ethidium bromide. Each PCR reaction corresponded to
the amount of DNA extracted from 5 × 105 cells.
Titrations of HTLV-1-positive (MT-2) and -negative (Jurkat) cellular
DNA were performed to determine sensitivity of the assay and detection
of as little as 0.05 ng MT-2 DNA (50 cells) per 500 ng Jurkat DNA was
achieved. We have previously determined that this MT-2 clone contains,
on average, 2.1 proviral copies per cell (data not shown). Thus, we
estimated the sensitivity of the PCR assay to be approximately 1 proviral copy per 5,000 cells. HTLV-1-specific products were confirmed
by digestion with Pst I which, in the wild-type sequence,
yielded fragments of 760 and 178 bp.
HTLV-1-specific PCR products were sequenced to further confirm
specificity and ensure absence of second site mutations within ORF
I/II. PCR reactions were purified (QIAquick; Qiagen) and sequenced by
automated dye terminator cycle sequencing method (ABI Prism; ABI,
Foster City, CA) using the primer pair 6555 and 7492, and an internal primer pair (6631, 5-GAGTCATCCCTGTAAACCAAGC-3; 7029, 5-AGAGGAAGCGAAAAAAAGAGCG-3).
For detection of the p12I message in transfected cells,
total mRNA was obtained by affinity column technique (RNeasy; Qiagen) and reverse transcribed using Moloney murine leukemia virus (MMLV) RT
(Boehringer Mannheim, Indianapolis, IN). Resulting cDNA
was amplified as described above using the primer pair RPX3 and
IK48 (corresponding to nucleotides 5094 and 6876, respectively), which spanned the second ORFI splice junction and thus
yielded products of 1,783 bp (corresponding to unspliced gag-pol and
singly spliced env transcripts) and 232 bp (corresponding to the doubly
spliced ORFI transcript). Primers that amplified a 183-bp transcript of the ABL gene were used as a control for amplifiable RNA.22
Rabbit inoculation procedures.
To compare the relative infectivity of HTLV-1-transfected human PBMC,
before inoculation we washed and plated 1 × 106 cells
of each transfectant culture in 24-well plates and maintained them in
complete RPMI for 72 hours. For inoculation we selected transfectants
that produced equal amounts of virus based on the amount of HTLV-1 p19
protein in the 72-hour culture supernatants by antigen capture assay at
the time of inoculation. Aliquots of all inocula were reserved for
analysis of total viral antigen by Western blot as previously
described,23 and mutation fidelity by PCR and RT-PCR as
described above.
Twelve-week-old specific pathogen free New Zealand White rabbits
(Hazelton, Kalamazoo, MI) were inoculated via the lateral ear vein with 1.0 × 107 gamma-irradiated (5,000 rad)
cells previously transfected with ACH, ACH.p12I, or vector
control (pKS). Initially, two rabbits each were inoculated twice at
12 and 25 weeks of age with either ACH- (R31, 32) or ACH.p12I-transfected PBMC (R33, 34) to ensure infectivity
of the inoculum. Subsequently, it was determined that a single
inoculation was sufficient to cause infectivity and a second group of
nine rabbits each were inoculated once at 12 weeks of age with either
ACH- (R51-R54), ACH.p12I- (R55-R58), or pKS- (R50)
transfected cells.
Serologic, clinical, and hematologic analysis.
Plasma antibody response to HTLV-1 in inoculated rabbits was determined
by use of commercial ELISA (Vironostika HTLV-1 Microelisa System;
Organon Technika, Durham, NC) that was adapted for use with rabbit plasma by substitution of alkaline phosphatase-conjugated goat anti-rabbit IgG (1:400 dilution) (Sigma). Plasma was diluted 1:12,800 (to obtain values in the linear range of the assay) and data
expressed as absorbance values. Reactivity to specific viral antigenic
determinants was detected using a commercial HTLV-1 Western blot assay
(Cambridge Biotech, Worcester, MA) adapted for rabbit
plasma by use of avidin-conjugated goat anti-rabbit IgG (1:3,000
dilution) (Vector, Burlingame, CA). Plasma showing reactivity to Gag (p24 or p19) and Env (p21 or gp46) antigens was
classified as positive for HTLV-1 seroreactivity.
Complete hematologic analysis was performed by automated cell counting
(Coulter, Hialeah, FL) and differential enumeration of
leukocytes and erythrocyte morphology in blood films. Body weights were
monitored and rabbits were regularly evaluated for any overt signs of
clinical disease. Rabbits were killed for necropsy and pathological
examination at postinoculation intervals of 12 (R50-R58) or 26 (R31-R34) weeks.
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RESULTS |
In vitro analysis of ACH and ACH.p12I transfectants.
To construct the ACH.p12I clone, a Pst I site at
ACH nucleotide 6732 corresponding to the splice acceptor for the last
exon of the ORF I message was deleted (PstI), resulting in
abrogation of the message encoding p12I (Fig 1). To confirm
that this message was not produced by ACH.p12I we
transfected ACH and ACH.p12I into human PBMC and analyzed
transfectant RNA by RT-PCR using primers RPX3 (5094) and IK4 (6876)
(Fig 1). The expected 230-bp product corresponding to the
p12I message was amplified from wild-type HTLV-1-infected
cells but not ACH.p12I-transfectants
(Fig 2A).
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| Fig 2.
Effect of p12I mutation on RNA, protein, and
infectious virus production in transfected PBMC. (A) RT-PCR of total
RNA from ACH- and ACH.p12I-transfecants, MT-2
(HTLV-1-positive) and Jurkat (HTLV-1-negative) cells. The 232-bp
p12I and constitutive 183-bp ABL transcript products are
indicated. (B) p19 antigen detected by ELISA in ACH and
ACH.p12I-transfectant PBMC culture supernatants. Results
are expressed as mean pg/mL ± SEM of p19 antigen obtained from 10 independent transfections. (C) HTLV-1 env-mediated syncytia formation
in HOS cells by ACH-, ACH.p12I-, or pKS- (vector control)
transfectants, Jurkat (HTLV-1-negative), or HuT102 (HTLV-1-positive)
cells. Results are expressed as mean number ± SEM of syncytia (four
or more nuclei) per well obtained from 10 wells (five independent
transfections); a, b, and c are significantly
different, P < .05. Note that HuT102 is a high
virus-producing cell line and is expected to induce higher numbers of
syncytia compared to the PBMC transfectants. (D) Infectious virus
production as measured by p19 antigen production from cultures
containing lethally irradiated ACH- or
ACH.p12I-transfectants alone or cocultured with
naïve rabbit PBMC. Results are expressed as absorbance values
for 1 and 7 day after wash culture supernatants measured by p19 antigen
ELISA.
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We then compared the mutant molecular clone ACH.p12I to
that of wild-type ACH for the ability to produce virus by measuring the
production of viral p19 matrix in transfectant culture supernatants by
ELISA (Fig 2B). No significant differences were seen in cell numbers or
amounts of p19 produced between ACH- or
ACH.p12I-transfectants, indicating that abrogation of the
ORF I message has no effect on viral core antigen production in vitro.
The PstI overlaps the env message in the noncoding region after the
env termination codon. To ensure that viral envelope expression was not
affected by the PstI, we measured envelope expression by monitoring
syncytia induction in HOS indicator cells after coculture with
transfected human PBMC. Coculture of HOS cells results in syncytia
formation and is dependent on expression of viral
envelope.21 No significant differences were seen in numbers
of syncytia induced in HOS cells by ACH- and
ACH.p12I-transfectants (Fig 2C).
To compare the in vitro infectivity of the wild-type and mutant clones
in rabbit primary cells, we cocultured lethally irradiated transfectants with naïve, activated rabbit PBMC. Viral p19
antigen was produced in similar amounts by all ACH- and
ACH.p12I-transfectant cocultures, but not in cultures
containing irradiated transfectants alone, suggesting that abrogation
of p12I has no effect on the ability of HTLV-1 to infect
rabbit PBMC in vitro (Fig 2D).
In vivo analysis of ACH and ACH.p12I transfectants.
To determine if abrogation of p12 affected the ability of HTLV-1 to
infect and replicate in rabbits, we inoculated six rabbits each with
ACH- or ACH.p12I-transfected, lethally irradiated human
PBMC. Before inoculation, there was no difference in the amount of
soluble p19 or cell-associated viral antigen produced by transfectants,
as measured by ELISA or Western blot analysis, respectively (data not
shown).
To ensure that the PstI was conserved in
ACH.p12I-transfectants at the time of inoculation, we
analyzed the HTLV-1 ORF I/II region in samples of the inocula. A 938-bp
region containing the mutation site was amplified from transfectants
using the 6555/7492 primer pair and then digested with Pst I. As expected, Pst I digestion of DNA amplified from
ACH-transfectants yielded fragments of 760 and 178 bp, in contrast to
that of ACH.p12I, which did not digest, since the
construction of the clone destroys the Pst I site coinciding
with ORF I splice acceptor (Fig 3). To
confirm these findings and to ensure that there were no second site
mutations within ORFs I or II, we sequenced the PCR products, which
include all of ORF I/II. Except for the expected PstI mutation, no
sequence differences were noted in ORFs I and II between the ACH- and
ACH.p12I-transfectants (data not shown).
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| Fig 3.
Conservation of PstI mutation in
ACH.p12I-transfectants at time of inoculation shown by PCR
amplification of genomic DNA from ACH-, ACH.p12I-, and pKS-
(vector control) transfected PBMC, MT-2 (HTLV-1-positive), and Jurkat
(HTLV-1-negative) cells. The native () or Pst I-digested (+) 938 bp HTLV-1 ORF I/II-specific product is present in
HTLV-1-positive cells and lacks the Pst I site corresponding to
the ORF I exon 3 splice acceptor site in
ACH.p12I-transfectants.
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To determine serologic response of rabbits to inoculated transfectants,
we measured anti-HTLV-1 antibody levels in rabbit plasma by ELISA.
Reactivity to specific HTLV-1 antigens was subsequently confirmed at
all time points by Western blot analysis. Data representing all
observed patterns of seroconversion are shown
(Fig 4). As expected after using infected
cellular inocula, seroconversion was detected in all rabbits as
indicated by rising antibody titers over the course of the experiment.
However, antibody titers were higher in ACH-inoculated rabbits (R51-55)
or rabbits receiving two inocula of either clone (R31-34) than in
rabbits receiving one inoculation of pKS- (R50) or
ACH.p12I- (R55-58) transfected cells (Fig 4A). The
intensity of Western blot reactivity correlated with ELISA titers and
all ACH- and ACH.p12I-inoculated rabbits were considered
seropositive for HTLV-1 (reactivity to gag and env antigens), although
rabbits inoculated once with ACH.p12I generally exhibited
weaker reactivity to a smaller number of HTLV-1 antigenic determinants
(Fig 4B).
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| Fig 4.
HTLV-1-specific serologic response in rabbits inoculated
with transfected PBMC. Represented are rabbits inoculated once (R50, 53, 57, and 58) or twice (R32 and 33) with ACH- (R53 and 32), ACH.p12-
(R57, 58, and 33), or pKS (vector control)- (R50) transfectants. Data
shown are absorbance values of plasma samples diluted 1:12,800 and
measured by anti-HTLV-1 antibody ELISA (A) or reactivity to specific
HTLV-1 antigenic determinants measured by Western blot analysis (B).
Note that reactivity of R50 (negative control) on ELISA is nonspecific
for HTLV-1 as shown by Western blot analysis and is attributable to
cross reactivity of cellular antigens in the inoculum and the ELISA.
The data shown for each rabbit are representative of the larger
inoculation group. Thus, R32 and R33 represent two rabbits each, R53
represents four rabbits, and R57 and R58 demonstrate two different
reaction patterns seen among the four rabbits comprising that inoculant
group.
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To determine the HTLV-1-infected status of inoculated rabbits, we
measured soluble p19 antigen in ex vivo PBMC culture supernatants (Table 1). Viral antigen was detected in
cultures from all ACH-inoculated rabbits by 6 weeks postinoculation.
However, we were unable to detect any p19 production in PBMC cultures
derived from ACH.p12I-inoculated rabbits at any time point.
As a further means of detecting infection in rabbits, we attempted to
amplify HTLV-1-specific proviral sequences from rabbit PBMC DNA by PCR.
Provirus was detected in all ACH-inoculated rabbits by 6 weeks
postinoculation (Table 1). In general, in ACH-inoculated rabbits,
HTLV-1-specific bands in comparison to -actin-specific bands were
faint or undetectable at weeks 1 and 2 and reached maximum intensity at
4 to 6 weeks, suggesting that recovery of provirus from these animals
was the result of replicating virus and not residual inoculum
(Fig 5). In contrast to the strong positive reactions in all ACH-inoculated animals, we were unable to consistently amplify proviral sequences from ACH.p12I-inoculated
rabbits, and when a positive reaction occurred the signal was very
weak. No ACH.p12-inoculated rabbits were positive for proviral
sequences at the termination of the study (Fig 5 and Table 1). These
data together with failure to detect antigen and weak serologic
responses in ACH.p12I-inoculated rabbits strongly suggest
that selective abrogation of p12I significantly interferes
with the ability of HTLV-1 to establish persistent infection in vivo.
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| Fig 5.
Detection of HTLV-1 proviral sequences in rabbits
inoculated with transfected PBMC. Genomic DNA from PBMC of rabbits
inoculated with ACH-(R51-54 and 31-32), ACH.p12- (R55-58 and 33-34), or
pKS (vector control)- (R50) transfectants was amplified by PCR using primers specific for HTLV-1 pX (938 bp) or -actin (415 bp).
Sensitivity of the PCR was estimated to be 1 proviral genome per 5,000 cells.
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DISCUSSION |
HTLV-1 p12I is a conserved 99-amino acid protein encoded by
an ORF located between the env and tax/rex genes.6-8 To
date the role of p12I in HTLV-1 replication has not been
identified, although in vitro overexpression of the protein suggests
interesting protein-protein interactions potentially affecting cellular
signal transduction or membrane receptor expression.7,8,10
Our findings indicate that abrogation of the p12I message
does not result in loss of viral infectivity. The destruction of the
ORF I splice acceptor site resulted in loss of expression of the
p12I message but had no effect on the ability of the
transfected virus to produce Gag or Env proteins, as measured by p19
ELISA and syncytia induction, respectively. Furthermore, we confirmed
that p12I-mutant virus is fully competent in the production
of infectious virus and that, like wild-type HTLV-1, this virus is
infectious in rabbit PBMC in vitro.
The ability of HTLV-1 to establish a persistent asymptomatic infection
in rabbits typical of the large majority of infected human beings is
well established.24 We and others have used rabbits as an
animal model of HTLV-1 infection to study viral transmission and test
potential vaccines.20,25-28 Here we used the rabbit model
to test the possibility that the effects of p12I ablation
would be manifested in vivo. At the time of inoculation into rabbits,
there were no differences in either cell number or amount of
replicating virus between ACH- and ACH.p12I-infected PBMC
inocula. The successful inoculation of these cells was confirmed by the
early seroconversion to HTLV-1 of all rabbits, with the exception of
the rabbit inoculated with pKS vector only. As in our previous studies,
ACH-transfected cells induced a vigorous and sustained humoral response
in rabbits against all major viral antigenic determinants. In contrast,
rabbits inoculated once with ACH.p12I exhibited a
relatively weaker humoral immune response. This was likely due to the
decreased or absent viral infectivity of the ACH.p12I
mutant compared with that of wild-type virus and is supported by our
inability to recover virus from ACH.p12I-inoculated
rabbits. The vigorous immune response noted in rabbits inoculated twice
with ACH.p12I can be attributed to the amnestic response
elicited by the second inoculation because, as in the other
ACH.p12I-inoculated rabbits, we failed to detect persistent
infection in these animals.
In asymptomatic HTLV-1-infected humans and rabbits, levels of viral
transcription are typically very low.26,29 Upon mitogenic activation of infected PBMC ex vivo, however, viral replication can be
efficiently induced.1,25 We were able to detect viral p19
antigen in 7-day PBMC cultures by 4 weeks in all ACH-inoculated rabbits. The absence of p19 antigen in most cultures from 1 and 2 weeks
postinoculation reflects the in vivo lag period of viral replication
and suggests that p19 antigen production was derived from newly
infected rabbit PBMC and not residual inoculum. Our inability to detect
p19 antigen from ACH.p12I-inoculated rabbits at any time
point suggests that viral replication was absent or depressed in
relation to wild-type virus.
In support of our previous findings that ACH is consistently infectious
in rabbits, we had no difficulty in amplifying HTLV-1-specific sequences from ACH-inoculated rabbits.17 As we noted with
ex vivo p19 antigen production, there was a period of 1 to 4 weeks in
these rabbits when we could not detect provirus by PCR, reflecting the
lag phase of viral replication. In contrast to ACH-inoculated animals,
we inconsistently detected provirus in ACH.p12I-inoculated
rabbits. Although occasional positive reactions were noted in these
animals, they were faint and at the lower limit of our assay
sensitivity, suggesting that proviral loads were much lower in rabbits
inoculated with the mutant clone. Furthermore, positive reactions from
ACH.p12I-inoculated rabbits were not consistent from week
to week, and all these rabbits were negative by the end of the study.
Although we cannot rule out that ACH.p12I-inoculated
rabbits were persistently infected with viral loads that were below our
limits of detection or that there exists in these rabbits a reservoir
of virus in an uninvestigated tissue or organ, our in vivo findings
provide strong evidence that HTLV-1 p12I is critical for
optimal viral infectivity and in maintaining the persistent
characteristic of the human infection.
Because we cultured transfected cells for 2 weeks before inoculation
into rabbits it was necessary for us examine the state of the PstI
mutation immediately before introduction of the cells into rabbits. If
the loss of p12I had an undetected, deleterious effect on
viral replication in vitro, there would be strong selective pressure
for rescue or reversion. However, no changes in the sequence of ORFs I
or II from that of wild type were detected in our inocula, and the
PstI mutation was preserved. The absence of any deleterious
phenotype in vitro makes the likelihood very small that mutations arose during the culture period in other genes required for viral
replication. Furthermore, if in vivo mutation of other viral genes were
to account for the observed phenotype, these would have had to occur independently against strong selective pressure in all six
ACH.p12I-inoculated rabbits but in none of the
ACH-inoculated animals. Thus, although this possibility cannot be
completely ruled out, we consider it extremely unlikely.
Ours is the first study to show a functional role for p12I
in viral replication or infectivity. It is not suprising that the functionality of this gene is only evident in vivo, as there are numerous examples of viral genes that are required only for efficient replication or pathogenesis in vivo, including simian immunodeficiency virus nef and herpes simplex virus 1 I34.5.30,31 The mechanism of the in vivo
functionality of p12I will require further studies to
elucidate. Because p12I has been shown to interact with the
IL-2R and chains, it may be speculated that modulation of the
IL-2 signaling pathway of HTLV-1-infected T cells by p12I
could enhance the transcriptional activity of the virus and promote replication.13,32,33 This effect, while potentially very
important for viral transmission in vivo, may be masked by the broad
activation state of transformed or mitogen and IL-2-treated primary
cells used in in vitro studies. Interactions of p12I with
other viral gene products may be important as well. Recently it has
been shown that proteins encoded by alternatively spliced mRNAs inhibit
the function of HTLV-2 Rex.34 Alternatively,
p12I may function to mask immune recognition of infected
cells through the downmodulation of cell-surface molecules such as
major histocompatibility complex gene products. Although
this association has not been shown, it is interesting to note that a
recent report suggests that p12I associates with immature
forms of IL2R- and chains, retaining them in the Golgi apparatus
and resulting in decreased cell-surface expression.13
Finally, it is important to note that our results do not rule out a
role of the untranslated p12I mRNA (eg, regulation of RNA
splicing) in the ability of the virus to establish an infection in
vivo.
HTLV-1 is a significant worldwide health problem and is known to cause
an aggressive T-cell malignancy.1 The identification and
characterization of p12I and other viral regulatory
proteins besides Tax and Rex will shed light on the mechanisms of
HTLV-1 replication and pathogenesis, as well as potential insight into
general mechanisms of oncogenesis. Furthermore, abrogation of
p12I may be an efficacious means of generating an
attenuated live-virus vaccine. Further studies will be necessary to
examine the plausibility of this idea and to elucidate the roles that
p12I plays in the life cycle of HTLV-1.
| |
FOOTNOTES |
Submitted November 6, 1997;
accepted February 4, 1998.
Supported by National Institutes of Health/National Cancer
Institute Grants No. CA-55185 (M.D.L.), P30CA16058 (OSU Cancer Center
core grant) and CA-63417 (M.D.L. subcontract from L.R.), and by
National Institutes of Health/National Institute of Allergy and
Infectious Disease Grant No. AI 01474 (M.D.L.) and the Glen Barber Fund
(N.D.C.).
Address reprint requests to Michael D. Lairmore, DVM, PhD, Department
of Veterinary Biosciences, The Ohio State University, 1925 Coffey Rd,
Columbus, OH 43210-1093.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Jason Kimata and Fen-Hwa Wong for construction of the
plasmids, Michael Robek for PCR primer design and helpful discussions, Patrick Green and Gary Kociba for critical reading of the manuscript, and Tim Vojt for preparation of figures.
| |
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