---
id: "edwards-2014-codependence"
title: "Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence"
authors:
  - "Cynthia A. Pise-Masison"
  - "Maria Fernanda de Castro-Amarante"
  - "Yoshimi Enose-Akahata"
  - "R. Cody Buchmann"
  - "Claudio Fenizia"
  - "Robyn Washington Parks"
  - "Dustin Edwards"
  - "Martina Fiocchi"
  - "Luiz Carlos Alcantara"
  - "Izabela Bialuk"
  - "Jhanelle Graham"
  - "Jean-Claude Walser"
  - "Katherine McKinnon"
  - "Bernardo Galvão-Castro"
  - "Antoine Gessain"
  - "David Venzon"
  - "Steven Jacobson"
  - "Genoveffa Franchini"
venue: "PLoS Pathogens"
year: 2014
date: "2014-11-06"
doi: "10.1371/journal.ppat.1004454"
url: "/research/publications/10-1371-journal-ppat-1004454/"
pdf: "/research/publications/10-1371-journal-ppat-1004454/dustin-edwards-10-1371-journal-ppat-1004454.pdf"
pmc: "https://pmc.ncbi.nlm.nih.gov/articles/PMC4223054/"
openAccess: true
license: "public-domain"
citedBy: 47
citedBySource: "OpenAlex, read 2026-09-12"
---
# Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence

HTLV-1 established infection in macaques only when it made both p12 and p8; making mostly one or the other failed.

## Abstract

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.

## Full text

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Co-dependence of HTLV-1 p12 and p8 Functions in Virus
Persistence
Cynthia A. Pise-Masison1, Maria Fernanda de Castro-Amarante1, Yoshimi Enose-Akahata2,
R. Cody Buchmann1, Claudio Fenizia1, Robyn Washington Parks1, Dustin Edwards1, Martina Fiocchi1,
Luiz Carlos Jr Alcantara3, Izabela Bialuk4, Jhanelle Graham2, Jean-Claude Walser5, Katherine McKinnon6,
Bernardo Galva˜ o-Castro3, Antoine Gessain7, David Venzon8, Steven Jacobson2, Genoveffa Franchini1*
1 Animal Models and Retroviral Vaccines Section, National Cancer Institute, Bethesda, Maryland, United States of America, 2 Viral Immunology Section, Neuroimmunology
Branch, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, United States of America, 3 Oswaldo Cruz Foundation Salvador, Bahia, Brazil,
4 Department of General and Experimental Pathology, Medical University in Białystok, Białystok, Poland, 5 Evolutionary Biology, Genetic Diversity Centre, University of
Basel, Basel, Switzerland, 6 Vaccine Branch Flow Cytometry Core Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of
America, 7 Unite´ d’Epide´miologie et Physiopathologie des Virus Oncoge`nes, De´partement de Virologie, Batiment Lwoff, Institut Pasteur, Paris, France, 8 Biostatistics and
Data Management Section, National Cancer Institute, Bethesda, Maryland, United States of America
Abstract
HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-
infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8.
Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced
expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral
DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we
constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26).
As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated
into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those
animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral
DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in
THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-
cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1
clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in
Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed
by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral
persistence and spread require the combined functions of p12 and p8.
Citation: Pise-Masison CA, de Castro-Amarante MF, Enose-Akahata Y, Buchmann RC, Fenizia C, et al. (2014) Co-dependence of HTLV-1 p12 and p8 Functions in
Virus Persistence. PLoS Pathog 10(11): e1004454. doi:10.1371/journal.ppat.1004454
Editor: Charles R. M. Bangham, Imperial College London, United Kingdom
Received March 12, 2014; Accepted September 8, 2014; Published November 6, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting information files. The 1530 orf-I sequences for the HTLV-1 infected individuals are available from Genbank under the accession numbers in Text S1.
Funding: This work was supported by the Intramural Program at the National Cancer Institute, National Institutes of Health, Bethesda, MD. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: franchig@mail.nih.gov
Introduction
HTLV-1 causes Adult T-cell Leukemia/Lymphoma (ATLL)
[1,2] or HTLV-1 Associated Myelopathy/Tropical Spastic
Paraparesis (HAM/TSP) in approximately 2–3% of the 15–20
million individuals infected by the virus worldwide [3,4]. HTLV-1
persists in the host despite a vigorous cellular and antibody
response, suggesting that the virus has developed effective
mechanisms to counteract host immune surveillance [5]. The
HTLV-1 open reading frame-I (orf-I) protein products p12 and p8
increase NFAT activity [6,7], STAT-5 transcriptional activity
and IL-2 production [8–10] in T-cells. In addition, they cause
down-regulation of ICAM-1 and -2, but not ICAM-3 surface
expression, allowing escape of infected cells from NK cell killing
[11]. The p12 protein precursor is processed by proteolytic
cleavage that removes a non-canonical endoplasmic reticulum
(ER) retention/retrieval signal at its amino-terminus to yield p8
(Figure 1A) [12,13]. The p8 protein traffics to the cell surface, is
recruited to the immunological synapse following T-cell receptor
(TCR) ligation, and down-regulates TCR proximal signaling [14].
In addition, p8 increases cell adhesion and virus transmission and
is transferred to neighboring cells via cellular conduits [15]. Both
p8 and p12 can form homo- or hetero-dimers through a highly
conserved single cysteine (position 39) or are palmitoylated and
remain monomeric (Figure 1A) [16,17]. Orf-I knockout viruses are
not infectious in non-human primates [18], suggesting the impor-
tance of orf-I in human infection. Here, studying a cohort of 160
HTLV-1 infected individuals, using an experimental model of
PLOS Pathogens | www.plospathogens.org 1 November 2014 | Volume 10 | Issue 11 | e1004454

macaque infection and using in vitro relevant models of HTLV-1
infection, we demonstrate that natural mutations within orf-I can
affect the relative amounts of p12 and p8, which in turn, correlate
with viral DNA levels in blood, the best predictor of risk for the
development of HAM/TSP or ATLL [19–22]. In addition, we
demonstrate that both proteins are essential for the in vitro resistance
to cytotoxic T-lymphocyte (CTL) killing of HTLV-1 infected cells.
Results
HTLV-1 orf-I in humans
Analysis of orf-I was performed on 160 HTLV-1 infected
individuals from various geographical areas (Table 1), 79 had
HAM/TSP and 81 were carriers. Genomic DNA was isolated
from patient PBMCs and used to quantify the viral DNA load and
for studies on the orf-I gene. As expected, individuals with HAM/
TSP had significantly (p = 0.0001) higher PBMC viral DNA loads
than carriers (Figure 1B). We obtained DNA sequences for a total
of 834 clones from these patients and compared them to our
reference orf-I cDNA [13] and found 216 variants (i.e., one or
more nucleotide changes compared to the consensus sequence).
One hundred thirty of these variants (85%) were unique. The most
frequent non-synonymous mutations within the orf-I gene yielded
G29S, P34L, S63P, R88K, and S91P amino acid changes. In line
with orf-I being necessary for infection, none of the approximately
1600 orf-I sequences analyzed had a premature termination
codon. We selected 17 non-synonymous mutations based on either
their proximity to the cleavage sites or their frequency in humans
and inserted them into the reference orf-I cDNA (herein defined
as p12WT) and transfected the expression constructs into 293T-
cells. The relative amount of p8 and p12, evaluated by Western
blot and densitometric scans was calculated as a percentage of
total expression from the orf-I gene (Figure 1C). A minimum of
two up to 20 independent Western blot experiments were
performed as indicated for each mutant in the legend of
Figure 1C. We observed 3 distinct patterns of expression
(Figure 1C and Figure 1D). The first consisted of balanced
expression of p8 and p12, as for p12WT. All these mutations
were downstream of both cleavage sites (mutants S91P-61% of
patients, R88K-10% of patients, S69G-12% of patients and
R83C-6% of patients). The second class consisted of predominant
expression of p12 (mutants F3L-4% of patients, P45L-9% of
patients, S23P-15% of patients, P34L/F61L-23% of patients,
S63P-62.5% of patients, L66P-10% of patients and G29S-30% of
patients) (Figure 1A). The viral DNA loads for patients with the
G29S mutations (Supplemental Table S3) follow the same trend as
the overall patient pool in that patients with HAM/TSP had
higher viral DNA loads. The third pattern was generated by a rare
mutation in position 26 between the two cleavage sites from
aspartic acid (D) (present in p12 WT, see Figure 1A) to either
asparagine (N) (mutated in 5% of patients) or glutamic acid (E)
(mutated in 2% of patients), resulting in the predominant
expression of p8 [12].
Analysis of the three orf-I expression patterns and viral DNA levels
in blood revealed significantly higher viral levels in individuals whose
cDNA expressed both p8 and p12 (p = 0.05), compared to those that
predominantly expressed either p8 or p12 (Figure 1E). No correlation
with disease status was observed within this patient cohort.
p8 and p12 are required for persistent infection of
macaques
To directly assess the requirement of p8 and p12 on viral
infectivity and persistence, we engineered the HTLV-1 molecular
clone pAB [18], that carries an orf-I identical to p12WT
designated here as pAB-D26 (Figure 2A). Glycine 29 was
substituted with serine to generate pAB-G29S as this mutation
impairs cleavage of p12 to p8, resulting predominantly in p12
expression (Figure 1D) [12–18]. Because substitution of N or E at
position 26, results in predominant expression of p8 (Figure 1D),
we generated pAB-N26. Importantly, the mutations introduced in
the orf-I gene did not alter the amino acid sequences of the hbz or
orf-II genes that overlap with orf-I. The isogenic clone pAB-
p12KO, mutated at the orf-I initiation ATG to eliminate
expression of both p8 and p12 (Figure 2A) was used as a control
since it is infectious in vitro but not infectious in vivo [18]. The
molecular clones were co-transfected with an HTLV-1-LTR-
Lucifease construct into 293T-cells to demonstrate their equivalent
ability to produce the Tax protein and activate the viral LTR
(Figure 2B). All viruses produced equivalent amounts of intracel-
lular p24Gag (Figure 2C) and extracellular p19Gag (Figure 2D).
We generated stable 729.6 human B-cell lines producing the
viral mutants as described [18]. These cell lines were clonal and
expressed equivalent levels of intracellular p24Gag and extracel-
lular p19Gag (Figure 2E, lower panels). We observed differences
in viral transmission when the cell lines were co-cultured with the
reporter cell line, BHK1E6 [23], which contains the b-galactosi-
dase gene under the control of the HTLV-1-LTR promoter. The
D26, G29S, or 12KO viruses were transmitted equivalently, but
the N26 virus was transmitted 10-fold more efficiently (Figure 2E),
consistent with the ability of p8 to increase virus transmission [15].
Whether it is p8, p12, or both that contribute to the
requirement of orf-I for infection in vivo remains unclear [18].
To address this point, we inoculated intravenously the lethally c-
irradiated B-cell lines producing equivalent levels of p19Gag
(Supplemental Table S1) from the D26 virus into four macaques,
the N26 virus into four macaques and the G29S virus into four
macaques. One animal was inoculated with parental uninfected
729.6 cells as a control. Three (P834, P840, P872) of the four
animals exposed to the D26 virus became HTLV-1 positive by
PCR with viral levels greater than 50 copies per one million cells
for at least one time point throughout the study and fully
seroconverted for viral antigens (Figure 3). In contrast, only one
animal (P845) exposed to the N26 virus was PCR positive for viral
DNA and only three animals showed weak reactivity to HTLV-1
antigens. None of the four animals in this study or four animals
from a previous study exposed to G29S virus became PCR positive
or seroconverted (Figure 3). We verified that the virus in animal
Author Summary
HTLV-1 persists despite a vigorous host immune response.
We found that polymorphism of HTLV-1 orf-I alter the
relative amounts of the p12 precursor and its cleavage
product p8, and is associated with differences in blood
virus levels in humans, a correlate of disease risk. Reverse
genetics in 160 HTLV-1 infected individuals demonstrated
that equivalent levels of p8 and p12 are associated with
high virus levels and, accordingly, genetically engineered
HTLV-1s that express either predominantly p12 or p8 are
poorly infectious in macaques. We found that expression
of p8 is sufficient for productive infection of monocytes.
Expression of either p12 alone or p8 alone is insufficient to
protect infected cells from MHC-class-I restricted CTL
killing. However, the balanced expression of both provides
resistance of infected cells to CTL killing. Together, our
findings provide the rationale to explore novel approaches
to target the cleavage of the p12 protein, an essential step
for viral infectivity and persistence.
p12 and p8 Functions in Virus Persistence
PLOS Pathogens | www.plospathogens.org 2 November 2014 | Volume 10 | Issue 11 | e1004454

P845, infected with N26 virus, retained the mutation at position 26
by cloning and sequencing orf-I from its PBMCs. These results
suggest that expression of both p12 and p8 is required for efficient
HTLV-1 infection and viral persistence. However, they also
suggest that p8 may be sufficient for infection and at least partial
seroconversion; particularly since none of the eight animals
inoculated with virus predominantly expressing p12 seroconverted
or had detectable viral DNA.
p8 is essential for productive infection of monocytes
HTLV-1 infects monocytes and dendritic cells [24–26] but the
role of infected monocytes to HTLV-1 pathogenesis remains
unclear. We have previously demonstrated that the abrogation of
orf-I expression results in loss of HTLV-1 infectivity of primary
monocyte-derived dendritic cells [18] and further that infection of
the monocytic cell line THP-1 mirrored results of ex vivo, primary
dendritic cells [25]. To define the relative contribution of p8, p12,
Figure 1. Analysis of
orf-I from the PBMCs of HTLV-1 infected individuals. (A) Schematic diagram of the Orf-I protein. The non-canonical
endoplasmic reticulum (ER) retention sequence is underlined by a solid bar. Black arrows indicate the putative cleavage sites, as well as the start of
the p8 isoform. Mutations which identify cleavage variants at position 26 and 29 are indicated in bold below the sequence. (B) Comparison of viral
DNA levels in PBMCs from HTLV-1 infected individuals by disease association, HC: healthy carrier (open symbols) and HAM/TSP: HTLV-1 associated
myelopathy/tropical spastic paraparesis (filled symbols). The data from 70 healthy carriers (HC) (n = 70) and 66 HAM/TSP individuals (n = 66) were
analyzed using the Mann-Whitney Test stratified by disease status. The statistically significant difference is marked with the p value. The horizontal
lines represent the mean viral DNA load. (C) Cloned orf-I cDNA constructs were transfected into 293T-cells and protein expression analyzed 48 hours
after transfection. The density of p12 and p8 bands was measured using AlphaView Software on an AlphaImager (ProteinSimple, San Leandro, CA).
Expression of p12 and p8 were added to give 100% expression. The percent of total Orf-I expression for each clone was graphed. The black bars
represent the percentage of p12 expressed and the lighter bar represents the percentage of p8 expressed. The clone is indicated at the bottom of the
graph. Expression patterns for each clone were examined in independent transfection experiments where n = 20 for D26, n = 8 for G29S; P45L, n = 7
for P34L/F61L, n = 6 for S69G; S23P; S63P; D26E; P34L, n = 5 for C39R/L40F/R83C; F3L; L66P; D5-L6M, n = 4 for R83C, D26N, n = 3 for S91P, n = 2 for
R88K; S63P/S91P. The expression patterns could be divided into three groups: p12 and p8, p12 mainly (p12) or p8 mainly (p8). (D) Representative
western blot analysis of cell lysates for Orf-I expression, using anti-HA (upper panel) or a loading control (anti-tubulin, lower panel) was performed.
Amino acid changes are indicated above each lane. The p12 or p8 isoform is indicated by arrows at the right. (E) Viral DNA levels in PBMCs from
individuals with the indicated orf-1 gene expression patterns are indicated in the x-axis. The data obtained was a total of 136 individuals using the
same assay (n = 10 individuals with mainly p8 expression, n = 32 individuals with mainly p12 expression and n = 94 individuals with similar p12 and p8
expression) and analyzed by an exact Wilcoxon rank sum test stratified by disease status. The horizontal lines represent the mean viral DNA levels.
The open symbols identify healthy carriers and the filled symbols HAM/TSP patients. The statistical significance is indicated by the p value.
doi:10.1371/journal.ppat.1004454.g001
p12 and p8 Functions in Virus Persistence
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or both to monocyte infection, we exposed the monocytic cell line
THP-1 to equivalent amounts of virus as measured by p19Gag
from unfiltered cell-free supernatants. Representative cultures are
shown (Figure 4). Cultures exposed to D26 or N26 viruses had
greater than 10,000 pg/ml of p19Gag in their supernatants at
week 2 (Figure 4A) and virus production was maintained up to 16
weeks. In contrast, cultures infected with G29S or 12KO viruses
had only background levels of p19Gag, as seen in control cultures
(mock-infected with 729.6 culture supernatant). Genomic DNA
isolated from the exposed THP-1 cells at week 18 was tested by
nested PCR for viral DNA. The level of HTLV-1 DNA detected
by PCR was consistent with the level of p19Gag released into the
supernatant and was highest in the cultures infected with the D26
and N26 viruses (Figure 4B). Quantitative PCR showed that the
D26 and N26 infected cultures contained 3–4 viral DNA copies
per cell, while the G29S and 12KO infected cultures contained
less than 1 copy per cell (Figure 4C). Interestingly, despite the
differences in viral production, all HTLV-1 infected THP-1
cultures displayed down-regulation of CD14 and up-regulation of
the activation markers HLA-DR and CCR7 (Figure 4D). These
results suggest that p8 expression is necessary and sufficient for
productive HTLV-1 infection in monocytes since p8 is expressed
in both D26 and N26, but not in G29S and 12KO.
Evasion of CTL activity requires both p12 and p8
CTLs play an important role in limiting viral replication and
spread by recognizing and lysing virally infected cells. The orf-I
protein products interfere with the normal trafficking of the MHC-
class-I molecule and are thought to reduce CTL recognition
[27,28]. To dissect the impact of p12 and/or p8 on CTL responses
in the context of the whole virus, we generated immortalized
infected CD4+ T-cells lines from an HLA.A2 healthy donor that
allowed the use of the human CTL clone from a HAM/TSP
patient that recognize the HLA.A2 restricted Tax peptide [11–19]
[29]. The CD4+ T-cells were cultured for over a year prior to
analysis; viral production, viral DNA copy numbers, and the level
of expression of the orf-I gene in the infected cultures is
summarized in Supplementary Table S2. In line with previous
results [15], T-cells producing N26 transmitted virus better than
those producing D26, G29S and p12KO (Figure 5A).
We reported previously that orf-I expression down-regulates
the surface expression of major histocompatibility complex
(MHC)-class-I in overexpression models [28]. Interestingly, in
here, we observed that the surface expression of HLA.A2 was
clearly down-regulated in CD4+T-cells that produce the G29S
virus indicating that p12 expressed by the virus, down-regulates
MHC-class-I in primary human CD4+ T-cells (Figure 5B).
Next, we studied whether infection of T-cells with the different
viruses affected their susceptibility to CTL killing. The CD4+ T-
cells infected with D26, N26, G29S and 12KO were loaded with
equivalent amounts of the immunodominant Tax [11–19]
peptides and co-cultured with the CTL clone at various effector-
to-target ratios. We observed the highest CTL killing of the 12KO
cells, suggesting that the absence of both p8 and p12 makes cells
susceptible to CTL killing (Figure 5C). We found reduced killing
of cells infected with N26 that predominantly express p8, as well as
in G29S that predominantly express p12. Strikingly, we observed
nearly complete resistance to CTL killing at all effector-to-target
ratios of cells infected with D26 that express a balanced level of p8
and p12 (Figure 5C). The resistance to CTL killing of the D26
infected CD4+ T-cells was abrogated by siRNAs targeting the orf-I
mRNA but not control siRNA (Figure 5D and 5E). These results
suggest that balanced expression of p12 and p8 is required to
protect HTLV-1 infected cells from CTL killing.
Discussion
The p12 precursor, encoded by orf-I, contains two proteolytic
cleavage sites, the first site, between amino acids 9 and 10 and the
second site, between amino acids 29 and 30 [12]. The p12
precursor is an ER associated protein and its cleavage removes a
non-canonical ER retention/retrieval signal that generates p8, a
protein that localizes to the cell surface [12]. Both p8 and p12
interact with the b and cc chains of the interleukin-2 receptor (IL-
2R) [10], the heavy chain of the MHC-class-I [28], calreticulin
and calnexin [30], and ICAM-1 and ICAM-2 [11]. The p8
protein traffics to lipid rafts, is recruited to the immunologic
synapse following T-cell receptor (TCR) ligation where it down-
regulates TCR proximal signaling [12] and co-localizes with
lymphocyte function-associated antigen-1 (LFA-1), increasing its
clustering [15]. The p8 protein also increases T-cell adhesion, the
formation of cellular conduits, and HTLV-1 transmission [15]. A
novel feature of p8 is its ability to be rapidly transferred from cell-
to-cell through cellular conduits [15]. Here, we investigated the
Table 1. HTLV-1 patients.
Region Clinical Status Patients Number Total
South America HAM/TSP 38 83
Carrier 45
Africa HAM/TSP 3 12
Carrier 9
North America and HAM/TSP 23 40
Caribbean Carrier 17
Asia HAM/TSP 1 2
Carrier 1
Unknown origin HAM/TSP 14 23
Carrier 9
Total 160
The orf-I sequence from 160 HTLV-1 infected individuals, from the indicated geographical regions, were evaluated. The patients were grouped by clinical status as either
an HTLV-1 carrier or HAM/TSP.
doi:10.1371/journal.ppat.1004454.t001
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specific contribution of each isoform to viral infectivity of T-cells
and monocytes in vitro and in viral persistence in vivo.
We hypothesized, that genetic mutations, which surround the
putative cleavage sites, affect the relative levels of p8 and p12 that
may have consequences in HTLV-1 infection. By using reverse
genetics on samples from HTLV-1 infected individuals, we have
identified genetic polymorphisms that affect the efficiency of
cleavage of the p12 precursor protein into p8. The first and most
frequent group of mutations results in an intermediate efficiency of
cleavage that yields an equivalent amount of p8 and p12. This
phenotype is associated with productive infection of monocytes
and a high viral DNA level in blood that is a correlate of disease
development [20–22,31]. The second most frequent phenotype,
yields predominantly p12 and affects the ability of the virus to
productively infect monocytes. The third rarer phenotype, results
in higher levels of p8 and a virus that retains its ability to
productively infect monocytes. However, consistent with our
studies in macaques and CTL sensitivity, both the second and
third phenotypes are associated with low virus DNA levels in blood
of naturally infected humans. We found that HLA-DR expression,
as well as CD80 and CCR7 were up-regulated in monocyte
cultures infected by HTLV-1, even when low or undetectable viral
proteins are expressed. The role of infected monocytes to HTLV-1
pathogenesis is unclear. However, HTLV-1 infection of mono-
Figure 2. Mutant viruses produce equivalent levels of Gag protein but the virus N26 is transmitted better. (A) The schematic diagram
of the HTLV-1 molecular clones indicates the amino acid change in each clone. The initiation codon for Orf-I is mutated in p12KO such that no Orf-I
protein is made. The changes did not affect the sequence and/or function of the overlapping pX region genes. Infectious molecular clones or control
DNA were co-transfected with an HTLV-1-LTR-luciferase construct and the renilla-luciferase transfection efficiency control into 293T-cells and culture
supernatants or protein lysates prepared 48 hours after transfection. (B) The HTLV-1 promoter activity induced by the HTLV-1 mutant was measured
by assaying luciferase activity from transfected cell lysates. Luciferase activity for each clone (indicated on the x-axis) from three independent
transfection experiments was graphed (n = 3). LTR-luciferase activity was normalized using the transfection efficiency control renilla-luciferase activity.
Error bars indicate the standard deviation. (C) Western blot analysis of protein lysates from transfected cells was assayed for intracellular p24Gag
expression (top panel) or the loading control, tubulin (bottom panel). (D) Culture supernatants from transfected 293T-cells were collected, spun to
remove debris and assayed for p19Gag levels using an HTLV-1 ELISA kit. The values graphed are from three independent experiments (n = 3). (E)
Stable producer 729.6 B-cell lines were cloned and used to quantify the transmission of the viral mutants. The 729-HTLV-1-producing cells or parental
control cells were co-cultured with BHK1E6 cells and 48 hours later, adherent cells were stained for -galactosidase activity. Graphed is the number of
blue cells per well for the indicated clone from three independent wells (n = 3). Error bars indicate standard deviation. By ANOVA and t- test,
transmission of WT, D26N and G29S was significantly different than control (p,0.0001). Transmission of D26N was significantly different than WT,
G29S and p12KO (p = 0.0007). There was no significant difference among transmission of WT, G29S and p12KO. Western blot analysis for HTLV-1
p24Gag was performed on whole cell extracts from 729-HTLV-1 producing cell lines. The housekeeping gene tubulin is shown for a loading control
(lower panels).
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cytes has been demonstrated [25,26,32,33] and viral infection is
associated with an increased frequency of more differentiated
monocytes (CD16bright) that may spread the virus to tissues [34].
These results suggest that orf-I plays a role in viral persistence
however, an early study by Furukawa and colleagues [35] found in
one HAM/TSP patient a virus with a mutation at the start codon
of orf-I and that this virus was transmitted in the individual’s
family. In contrast to our work, Furukawa et al. did not clone the
orf-I products and assess its expression and stability [35]. In
addition, the authors have not ruled out that the orf-I gene was
expressed in those individuals through alternative splicing. Several
groups have shown that cryptic splice sites and donor sites are
present in retroviral sequences and that gene products can be
produced through alternative splice acceptor/splice donor usage.
Thus, although we have not demonstrated an absolute require-
ment for orf-I in HTLV-1 infection in humans, it is clearly
required in non-human primates [18]. In addition, the results of
this study and that of Furukawa et al. [35], finding only 1 in 304
patients which do not retain orf-I expression (0.3%), suggests that
orf-I expression is likely to provide an advantage in HTLV-1
persistence.
Over-expression studies showed that p12 contributes to evasion
from CTL by interacting with the MHC-class-I Heavy chain (Hc)
in the ER and preventing its association with b2-microglobulin
[27,28,36]. This interaction induces the MHC-class-I Hc retro-
translocation into the cytosol for degradation by the proteasome,
decreasing cell surface MHC-class-I. The p8 protein was recently
shown in exogenous expression studies to be transferred to
uninfected cells [15]. Therefore, we speculate that the contribution
of p8 to CTL escape may be ascribed to the ability of this protein to
be transferred to CD8+ T-cells, whereby it may down-regulate TCR
signaling, resulting in the weakening of the strength of the
immunological synapse [15], and inhibition of CTL degranulation.
Indeed, the p8 protein is recruited to lipid rafts within the
immunological synapse upon engagement of TCR by CD3
ligation and causes T-cell anergy [12]. More recent studies
demonstrated a reduction in the strength of the immunological
synapse in the presence of p8 [15]. This is in line with the
finding that not only the number of HTLV-1-specific CTLs is
important, but also their functional avidity [37] and even if
they are abundant [38–40], they do not clear infection.
Collectively our results suggest a model whereby a combina-
tion of effects of p8 and p12 on monocyte infectivity, viral
transmission, and escape from CTL favors viral persistence
(Figure 6).
A virus expressing both p12 and p8 (D26) infects monocytes,
is efficiently transmitted to CD4+ T-cells, renders them less
prone to CTL lysis and persists (Figure 6A). In contrast, a virus
ablated in p8 and p12 expression (12KO) is poorly infectious in
monocytes and CD4+T-cells in vitro, the infected cells are
susceptible to CTL killing and infection is not sustained in vivo
(Figure 6B). Virus expressing mainly p8 (N26), has an interme-
diate phenotype; it maintains its infectiousness for monocytes
and CD4+ T-cells, but because it only partially protects infected
CD4+ T-cells from CTL does not cause a robust infection in
vivo (Figure 6C). Consistent with the concept of co-dependence
of p8 and p12 functions for viral persistence in the host, a virus
expressing mainly p12 (G29S) is poorly infectious in monocytes,
the CD4+ infected T-cells are partially susceptible to CTL
killing and the virus is not infectious in macaques (Figure 6D
and Figure 3).
It is likely that p12 and p8 also affect other steps in antigen
processing and presentation of HTLV-1 peptides on MHC-class-I.
The p12/p8 proteins interact with calnexin and calreticulin [30]
which may affect the folding of MHC-class-I and its peptide
loading [41]. Similarly, the interaction of p12 and p8 with the
16 kDa protein of the V-ATPase that occurs in the ER [42] may
prevent the assembly of the mature form of the V-ATPase and
acidification of the secretory pathway. Recent work shows that the
p8 protein traffics to the cell surface via the secretory pathway
[12]. Thus, p8’s association with the V-ATPase could alter not
only the secretory and the endocytic pathway, but also receptor
recycling on the cell membrane. Thus, future work is necessary to
assess whether p12 and p8 interaction with the V-ATPase has
important functional implication in antigen processing and
presentation.
Figure 3. D26, N26, and G29S infectivity in macaques. Sera from inoculated male Rhesus macaques were assayed for reactivity to HTLV-1
antigens. The animal number and inoculation group are indicated above each sample. Indicated below each western blot strip is the time of sera
collection. The presence of HTLV-1 viral DNA was measured from PBMC DNA isolated at the designated time points by PCR analysis for HTLV-1
integrase; (-) indicates PCR negative. Viral DNA loads were normalized to the macaque albumin gene and expressed as the number of HTLV-1 viral
DNA copies per 106 PBMCs. The value of the viral DNA load provided at the bottom of the figure is the highest measured for the indicated animal.
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Our results from the macaque studies and CTL killing assays
suggest that HTLV-1 virus expressing p12 only should be
efficiently eliminated. However, we do find infected individuals,
both carriers and HAM/TSP patients, harboring HTLV-1-p12
only virus. Several factors may influence the persistence of
HTLV-1 p12 only virus. First, from earlier studies by Nicot et al
[8], p12 expression through its activation of STAT5 decreases
the IL-2 requirement and thus confers a proliferative advantage
to infected cells. Second, we see that expression of p12 alone
does down-modulate MHC class 1 expression on infected T-
cells. The down-modulation could be sufficient in vivo to allow
escape from some CTL clones. Third, p12 protein has been
shown to down-modulate ICAM-1 and ICAM-2 suggesting that
infected cells would be less susceptible to NK cell killing [11].
Further, while we find that HTLV-1 G29S virus does not
productively infect THP-1 cells, infection does occur and in
preliminary studies we find that activation of infected cells
stimulates infectious virus production. This would allow the
virus to persist undetected in an infected individual and upon
activation spread of the virus. Finally, from our studies on p13
and Tax [43] we find that there is significant interplay between
viral proteins. Our studies have focused on orf-I mutations, but
it is possible that changes in other viral genes can impact the
role of orf-I in immune evasion.
In conclusion, our data suggest that while infection of
monocytes is important in HTLV-1 infection, viral persistence
also necessitates a coordinated expression of p12 and p8 to
avoid CTL recognition of infected cells. Thus, pharmacolog-
ically altering the efficiency of cleavage of the p12 precursor
could have profound effects on viral persistence, by restoring
the effectiveness of the host immune response to HTLV-1
and ultimately decreasing the risk of disease development
through the reduction of the number of HTLV-1 infected
cells.
Figure 4. HTLV-1 infection of the monocytic cell line THP-1. (A) THP-1 cells were infected with supernatants from 729-HTLV-1 producing or
parental 729.6 cell lines (concentrated by ultracentrifugation). Culture supernatants were monitored by ELISA for p19Gag levels. Graphed is the log
scale of p19Gag in picograms per milliliter over a 16 week period for one set of cultures. THP-1 infected cultures: D26 (white bar); N26 (black bar);
G29S (slanted bar); 12KO (dotted bar); Mock (gray bar). The dashed line indicates assay background level. (B) PCR analysis was performed on genomic
DNA isolated at week 16. The first (upper panel) and second (lower panel) rounds of nested PCR were separated by electrophoresis and stained with
ethidium bromide to visualize products for the indicated cell cultures. Arrows designate the Gag and the control b-actin fragments. (C) The viral DNA
copy number for each cell culture at week 18 was determined by quantitative real-time PCR. The human albumin gene was used for normalization.
(D) Histogram plots show the phenotype of HTLV-1 infected THP-1 cells for the cell surface monocytic markers: CD14, HLA-DR and CCR7. Each viral
mutant (gray line) was compared to the wild-type (D26, un-shaded, black line) and the mock (shaded) infected THP-1 cells.
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Materials and Methods
Ethics statement
This study was carried out in strict accordance with the
recommendations described in the Guide for the Care and Use of
Laboratory Animals of the National Institute of Health, the Office
of Animal Welfare and the United States Department of
Agriculture. All non-human primate work was approved by the
NCI Division of Intramural Research Animal Care and Use
Committees (IACUC; protocol no. 458). The animals were
housed, feed, given environmental enrichment and handled in
accordance with the standards of the Association for the
Assessment and Accreditation of Laboratory Animal Care
International. Appropriate steps were taken to minimize suffering
in accordance with the Weatherall report (‘‘The use of non-human
primates in research’’). The animals were housed and experiments
conducted at Advance Bioscience Laboratories in Rockville, MD
in accordance with the standards of the American Association for
Accreditation of Laboratory Animal Care. Non-human primates
are housed in a rolling rack system and the cage configuration
within the rooms allow for establishment of visual contact with
other species members. Positive human interaction with the staff
includes providing food treats, positive verbal and non-verbal
communication, systematic husbandry and consistent staffing. A
dietary enrichment and novel food program has been in place in
the colony since 1987. Each animal is provided with sensory and
cognitive enrichment that include foraging and food-based
enrichment strategies, toys, auditory and visual enrichment and
Figure 5. Susceptibility of HTLV-1 producing CD4+ cell lines to CTL killing. (A) CD4+ T-cells infected with the D26, N26, G29S and 12KO
viruses were incubated with BHKE16 indicator cells for 48 hours. Un-infected Jurkat T-cells (control) were used as a negative control. The number of
blue cells per well for three independent experiments is graphed (n = 3). Error bars indicate standard deviation. (B) A comparison of the surface
expression of CD4 (left panels) and HLA.A2 (right panels) are shown for the indicated virus-infected CD4+ T-cells (black line) in comparison to the
12KO CD4+ T-cell line (shaded). (C) Cytoxic T-lymphocyte killing assays were done to evaluate specific lysis of CD4+ T-cells infected with the D26, N26,
G29S and 12KO viruses. A long term HLA.A2 restricted CD8+ T-cell line from an HAM/TSP patient was used as the effector cell (see Materials and
Methods). Graphed is the percent of specific lysis at varying effector-to-target cell ratios (1.25:1; 5:1; 20:1). The graph represents data from at least two
independent experiments done in triplicate (n$2). Lysis of 12KO at the 20:1 ratio (highest specific lysis) was set to 100%. All samples were normalized
to maximal killing obtained with the 12KO virus. Error bars indicate standard deviation. (D) Western blot analysis of protein lysates from transfected
cells was assayed for p12 and p8 expression (top panel) or the loading control, tubulin (bottom panel) to determine the effectiveness of the siRNA
constructs. Cells were co-transfected with p12WT cDNA or control expression constructs in the presence or absence of siRNA (Si Ctrl or Si orf-I). (E)
CD4+ D26-infected cells were transfected with siRNA control (Si CTRL) or siRNA to orf-I (Si orf-I) and used as target cells in CTL killing assays. CTL lysis
of the cells at increasing effector-to-target cell ratios is graphed. The graph represents data from at least two independent experiments done in
triplicate (n$2). Error bars indicate standard deviation.
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hideaways. All procedures were carried out under anesthesia
(Telazol, Ketamine/Xylazine or Ketamine HCl) by trained
personnel under the supervision of veterinary staff and all efforts
were made to ameliorate the welfare and to minimize animal
suffering in accordance with the Weatherall report for the use of
non-human primates recommendations. Early endpoint criteria,
as specified by the IACUC approved score parameters, were used
to determine when animals should be humanely euthanized.
Blood samples from HTLV-1-infected patients and non-infected
(ND) donors were obtained from the Centre Hospitalier Uni-
versitaire de Fort-de-France in Martinique and Institut Pasteur de
Cayenne in French Guyana, the Bahia School of Medicine and
Public Health and the National Institutes of Health Clinical
Center. Patients suffering from HAM/TSP or HTLV-1 asymp-
tomatic carriers were recruited according to World Health
Organization (WHO) criteria. All subjects gave fully informed,
written consent and all clinical investigations have been conducted
according to the principles expressed in the Declaration of
Helsinik. All samples were anonymized and research conformed
to the guidelines of the ethics review board of the National Cancer
Institute.
Patient samples and HTLV-1 viral DNA loads
The study comprised 160 HTLV-1 infected individuals from
different geographical regions (Caribbean, France, North Amer-
ica, Africa, and Brazil) with different disease status (Table 1). The
subjects for the analysis were participants in research studies
conducted at the institutions of the authors. Informed consent was
written and obtained from each subject in accordance with the
Declaration of Helsinki. DNA extracted from PBMCs of HTLV-1
infected individuals was used to determine the viral DNA load.
Real-time PCR analysis of HTLV-1 (Tax) was performed with
100 ng of cellular DNA as previously described [19]. HTLV-1
viral DNA levels were calculated by the following formula: (copies
of HTLV-1 (pX)/(copies of beta-actin/2)6100 cells. We are using
the term viral DNA load since our assay does not distinguish
between integrated and unintegrated viral DNA. The same DNA
was used as templates for PCR reactions using Platinum High
Fidelity PCR Supermix (Invitrogen, Carlsbad, CA) according to
the manufacturer’s protocol. In the reaction, 10 pmol/ml of each
primer: 12-Fwd 59-CACCTCGCCTTCCAACTG-39, p12-p30-
Rev 59-GGAGTATTTGCGCATGGCC-39 were used for ampli-
fication of the p12-p30 (872 bp) region at Tm = 55uC. For samples
Figure 6. Model of p12 and p8 functions on monocyte, T-cell infection, and their susceptibility to CTL killing. The red dots represent
HTLV-1 virions/proteins and the solid arrows represent effective CTL killing of CD4+-infected T-cells. The dashed lines indicate no CTL killing. Lysed
cells are represented by misshapen, dashed lines. Cell types are indicated in the figure. D26-infected CD4+ T-cells expressing balanced levels of p12
and p8 (A); 12KO-infected CD4+ T-cells expressing neither p12 nor p8 (B); N26-infected CD4+ T-cells expressing mainly p8 (C); and G29S-infected CD4+
T-cells expressing mainly p12 (D).
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with no visible amplified PCR product 2 ml of the PCR reaction
was used as a template for nested PCR with primers: p12-nested-F
59-GTCTAGTATAGCCATCAACC-39 and p30-mid-nested-Rev
59- CTGGACAGGTGGCCAGTA-39. PCR products were puri-
fied by gel electrophoresis and QIAquick Gel Extraction Kit
(Qiagen, Valencia, CA) and subsequently, cloned into pCR4 TA
TOPO vector (Invitrogen, Carlsbad, CA) according to the
manufacturer’s protocol. QIAprep Spin Miniprep Kit (Qiagen,
Valencia, CA) was used for plasmids isolation. Five to 20 clones
per patient were isolated and sequenced. The 1530 orf-I sequences
for the HTLV-1 infected individuals are available from Genbank
under the accession numbers in Text S1. The study on the
immunophenotype of blood monocytes was performed on patient
samples obtained through the NIH Clinical Center (Table 1).
Expression plasmids
The pME18S p12deltaSL expression plasmid has been
described previously [14]. This plasmid served as a backbone for
generation of p12 mutants by means of PCR or by QuickChange
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using
site-specific mutagenic oligonucleotides according to the manu-
facturer’s instructions. The following oligonucleotides were used
and the sequence of plasmid clones was analyzed to confirm the
mutations.
F3L-F: 59-CCTAGCACTATGCTGCTTCGCCTTCTCAG-
CfCCCT-39
F3L-R: 59-AGGGGCTGAGAAGGCGAAGCAGCATAGT-
GCTAGG-39
S23P-F: 59-GCTCCTGCTCTTCCTGCTTCCTCCGGG-
CGACGTCAGCG-39
S23P-R: 59-CGCTGACGTCGCCCGGAGGAAGCAGGAA-
GAGCAGGAGC-39
D26N-F: 59-CCTGCTTTCTCCGGGCAACGTCAGCGG-
CCTTC-39 (for p12 subgroup A template – with S (serine) at
the 23rd amino acid position in p12)
D26N-R: 59-GAAGGCCGCTGACGTTGCCCGGAGAAA-
GCAGG-39 (for p12 subgroup A template)
D26N-F: 59 -CCTGCTTCCTCCGGGCAACGTCAGCG-
GCCTTC-39 (for p12 subtype B template - with P (proline) at
the 23rd amino acid position in p12)
D26N-R: 59-GAAGGCCGCTGACGTTGCCCGGAGGAA-
GCAGG-39 (for p12 subtype B template)
D26E-F: 59-CTGCTTTCTCCGGGCGAAGTCAGCGGC-
CTTCTTC-39
D26E-R: 59- GAAGAAGGCCGCTGACTTCGCCCGGA-
GAAAGCAG-39
G29S-F: 59-TGCTTTCTCCGGGCGACGTCAGCAGCCT-
TCTTCTC-39
G29S-R: 59-GCGGAGAAGAAGGCTGCTGACGTCGCC-
39
delta29-F: 59-GTGGCTCGAGACCATGCTTCTTCTCCG-
CCCGCCTC-39
delta29-R: 59-TCGGTCTAGAAACAACAACAATTGCATT-
CATTTTATGTTTCAGGTTCA-39
P34L-F: 59-GGCCTTCTTCTCCGCCTGCCTCCTGCG-
CCGTGC-39
P34L-R: 59-GCACGGCGCAGGAGGCAGGCGGAGAA-
GAAGGCC-39
P45L-F: 59-GCCTTCTCCTCTTCCTTCTTTTTCAAA-
TACTCAGC-39
P45L-R: 59-GCTGAGTATTTGAAAAAGAAGGAAGAG-
GAGAAGGC-39
S63P-F: 59-CTCCCGCTCTTTTTTCCGCTTCCTCTTC-
TCCTC-39
S63P-R: 59-GAGGAGAAGAGGAAGCGGAAAAAAGAGC-
GGGAG-39
L66P-F: 59-GCTCTTTTTTTCGCTTCCTCCTCTCCTC-
AGCCCGTCGCTGCCG-39
L66P-R: 59-CGGCAGCGACGGGCTGAGGAGAGGAGG-
AAGCGAAAAAAAGAGC-39
S69G-F: 59-GCTTCCTCTTCTCCTCGGCCCGTCGCTG-
CCGAT-39
S69G-R: 59-ATCGGCAGCGACGGGCCGAGGAGAAGA-
GGAAGC-39
R88K-F: 59-GGCTCTTTCTCCCCTGGAAGGCCCCGT-
CGCAGCCGGCCG-39
R88K-R: 59-CGGCCGGCTGCGACGGGGCCTTCCAGG-
GGAGAAAGAGCC-39
S91P-F: 59-CCCCTGGAGGGCCCCGCCGCAGCCGGCC-
GCGGC-39
S91P-R: 59-GCCGCGGCCGGCTGCGGCGGGGCCCTC-
CAGGGG-39. Expression of all mutants were assessed by western
blot analysis using the anti-HA1 antibody clones12CA5 and 3F10-
HRP (Roche Applied Science, Indianapolis, IN).
Cell culture and DNA transfection
293T- and BHK1E6 cells were grown in Dulbecco’s modified
Eagle medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 2 mM penicillin-streptomycin and 5 mM L-gluta-
mine. The 729.6 B-cells were grown in RPMI 1640 supplemented
with 10% FBS, 2 mM penicillin-streptomycin and 5 mM L-
glutamine. The HTLV-1 molecular clones pAB-D26 (WT), pAB-
G29S (p12), and pAB-p12KO were previously described. To
generate pAB-N26 (p8), mutation of GAC to AAC at amino acid
26 of orf-I (a glutamic acid to asparagine substitution) was
introduced into the pBST ClaI/SalI cassette using QuickChange
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and then
ligated to the pACH backbone. The mutant clones were verified
by DNA sequencing of the ClaI/SalI fragment inserted in the
provirus.
To confirm that the clones were producing virus, they were
transfected into 293T-cells using Effectene reagent (Qiagen,
Valencia, CA). Briefly, 10 mg of DNA of pAB-D26 (WT), pAB-
N26, pAB-G29S, and pAB-p12KO was transfected into 10 cm
dish of 293T-cells. After 48 hours, the cells were extracted for total
protein with radioimmunoprecipitation assay (RIPA) buffer and
analysis of intracellular HTLV-1-p24 (Advanced BioScience
Laboratories Inc., Rockville, MD) and tubulin (Sigma-Aldrich,
St. Louis, MO).
Intracellular Tax expression was characterized by co-transfect-
ing molecular clones and an HTLV-1-LTR-luciferase reporter
into 293T-cells. The pRL-TKLuc plasmid was used as a
transfection control. After 48 hours, cells were extracted with
Passive Lysis Buffer (Promega, Milwaukee, WI) and protein
samples analyzed with Dual-Glo reagent (Promega, Milwaukee,
WI) for LTR activation. The culture supernatant from these
transfections were spun down to remove any cell debris and
analyzed by p19Gag ELISA (ZeptoMetrix, Buffalo, NY) for virus
production.
The siRNA nucleofection assays were performed using the
Human T-cell Nucleofection Kit (Lonza, Basel, Switzerland) and
program O-017 as described by the manufacturer. Briefly, CD4+
D26 producing T-cells (26106) were incubated with 20 nM of
either control siRNA or siRNA to orf-I (59GCACUAUGCU-
GUUUCGCCUUCUCAG39) (Stealth RNA, Invitrogen, Carls-
bad, CA). Forty-eight hours after nucleofection, cells were used in
the cytotoxicity assay. Knockdown of Orf-I expression was
monitored by transient transfection of Orf-I expression constructs
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and siRNA into 293T-cells using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA) as described by the manufacturer.
Generation and characterization of HTLV-1 producing cell
lines
Stable HTLV-1 producing 729.6 human lymphoblastoid B-cells
were produced as described previously [18]. Briefly 729.6 cells
(56106) were electroporated with 5 mg of pAB-D26, pAB-G29S,
pAB-N26 or pAB-p12KO using AMAXA Nucleofector II,
Nucleofection kit V at M-013 (Lonza, Basel, Switzerland)
according to the manufacturer’s guidelines. Infected cells were
selected by culture in neomycin as previously described [44]. The
supernatant p19Gag production was measured by ELISA assay
(ZeptoMetrix, Buffalo, NY). To analyze the clonality of the cell
lines, 2–56107 cells were grown up in the absence of G418 and
genomic DNA extracted using a Genomic DNA Wizard Kit
(Promega, Milwaukee, WI) following the manufacturer’s guide-
lines. DNA (25 mg) was digested with either High Fidelity EcoRI
or XhoI (New England Biolabs, Ipswich, MA), run on an 0.8%
agarose gel overnight, denatured with Denaturing Solution
(Biosource, Madison, WI) and neutralized with Neutralizing Buffer
(KD Medical, Columbia, MD), then blotted overnight to Im-
mobilon NY+ membrane and crosslinked at 1.56106 J with a UV
crosslinker. Biotinylated probe was synthesized with a Phototope kit
and pAB-D26 molecular clone (New England Biolabs, Ipswich,
MA). Membrane was prehybridized in Ultrahyb buffer (Ambion
Life Technologies, Grand Island, NY) at 42uC for 2 hours, 10 pmol
of probe added and hybridized overnight. Membrane was washed
according to the Ultrahyb manufacturer guidelines and developed
using the Phototope Star chemiluminescence kit (New England
Biolabs, Ipswich, MA).
Using negative selection beads (Invitrogen, Carlsbad, CA),
CD4+ T-cells were isolated from un-infected peripheral blood
mononuclear cells. Stable HTLV-1 producing CD4+ T-cell lines
were established by co-cultivation of donor un-infected primary
HLA.A2+/CD4+ T-cells with lethally c-irradiated 729.6-HTLV-1
infected lines. T-cells were cultured in RPMI supplemented with
20% FBS and 100 U of interleukin-2 for one year. Virus
production was monitored by p19Gag ELISA (ZeptoMetrix,
Buffalo, NY) and viral genomic sequences verified by sequencing
of the ClaI-SalI fragment as described above.
Chronically infected THP-1 cells were produced as previously
described [25]. Briefly, supernatant from 729.6 HTLV-1
producer cell lines were collected and ultra-centrifuged at
23000 rpm for two hours and thirty minutes at 4uC to
concentrate the virus. Pellets were suspended in PBS and
p19Gag measured by ELISA assay (ZeptoMetrix, Buffalo, NY).
Equivalent amounts of p19Gag were used for infection of THP-
1 cells. Briefly, THP-1 cells were suspended in virus prepara-
tions and centrifuged at 3000 rpm for one hour at room
temperature in the presence of 8 mg/ml polybrene (Sigma, St.
Louis, MO). Cultures were maintained in RPMI 1640, 10%
FBS, with 50 mM b-mercaptoethanol and p19Gag production
monitored.
Infectivity assays
BHK1E6 cells (16105) containing a lacZ reporter gene
downstream of the,LTR promoter [23], were co-cultured for
48 hours with either control un-infected cells or HTLV-1
producers D26, N26, G29S and 12KO (16106). Monolayers were
washed twice with PBS to remove medium and suspension cells
and assayed using a b-galactosidase Staining Kit according
to manufacturer’s instructions (Active Motif, Carlsbad, CA). The
b-galactosidase expressing cells were counted by brightfield
microscopy.
Animal inoculation and sample collection
The macaques used in this study were male colony-bred Indian
Rhesus Macaques (RMs) obtained from Covance Research
Products. The animals were housed, feed, given environmental
enrichment and handled in accordance with the standards of the
Association for the Assessment and Accreditation of Laboratory
Animal Care International. Appropriate steps were taken to
minimize suffering in accordance with the Weatherall report
(‘‘The use of non-human primates in research’’). The care and use
of the animals were in compliance with all relevant institutional
(National Institutes of Health) guidelines. All macaques were 2–3
years of age and seronegative for simian T-cell lymphotropic virus
1 and simian immunodeficiency virus at the initiation of the study.
RMs were inoculated with lethally irradiated 729.6 producer cells.
Supernatant p19 levels were measured prior to inoculation and
cell numbers were adjusted to give equivalent amounts of p19Gag
per animal (Supplementary Table S1). Four macaques each were
used for the N26 and D26 viruses and eight each for the G29S
virus. One macaque was infused with irradiated control parental
729.6 cells. All macaques received an equivalent dose of virus
based on p19Gag expression levels per million cells, as previously
described [18].
Mononuclear cells were separated from whole blood specimens
by density gradient centrifugation (Ficoll). PBMCs (56106) from
animals positive for proviruses were washed in PBS and DNA
isolated using the Genomic DNA Wizard kit as described by the
manufacturer (Promega, Milwaukee, WI).
HTLV serology and quantitative PCR
Reactivity to specific viral antigens in the sera of infected
animals was detected with the use of a commercial HTLV-1
western immunoblot assay (GeneLabs Diagnostics, Redwood City,
CA). Quantitative Real-time PCR analysis was performed as
described previously [18]. Proviral loads were normalized to the
macaque albumin gene and expressed as the number of HTLV-1
proviral DNA copies per 106 PBMCs. The limit of detection for
the PCR assay is one copy in ten thousand cells. DNA sequencing
of the orf-I genes was performed from ex vivo samples of macaque
cellular DNA to check for reversions of point mutations to wild-
type. After genomic DNA was isolated from 56106 PBMCs, PCR
was performed using primers p12-Fwd, 59-CACCTCGCCTCC-
CAACTG-39 and p30-Rev, 59-GGAGTATTTGCG- CAT-
GGCC-39 which amplified the fragment (871 nucleotides)
spanning positions 6414 to 7285 of the HTLV-1 genome. The
PCR amplicon was cloned into the pCR4TOPO vector (Invitro-
gen, Carlsbad, CA) using the manufacturer’s protocol and 10
unique colonies sequenced. The level of orf-I mRNA was
quantified using splice site-specific quantitative RT-PCR (qRT-
PCR) as described [45]. After isolation of total cellular RNA, RT-
PCR was performed using primers: p12-1B, 59-GTCC-
GCCGTCTAG‘CACTATG-39; p12 reverse, 59-GGAGGAAG-
CAGGAAGAGC-39; probe, MP-1 59(FAM)-TTCGCCT-
TCTCAGCCCCTTGTCT-39(TAMRA). All samples were nor-
malized to Gapdh mRNA copy number.
Flow cytometry
Surface staining for CD4+ T-cells was performed for 30 minutes
at room temperature with antibodies to CD4 and HLA.A2 from
BD Biosciences (San Jose, CA). All cells were fixed with 1%
paraformaldehyde and at least 50,000 events acquired on an
LSRII (BD Bioscience, San Jose, CA). Data analysis was
p12 and p8 Functions in Virus Persistence
PLOS Pathogens | www.plospathogens.org 11 November 2014 | Volume 10 | Issue 11 | e1004454

performed with FlowJo 9.4 software (Tree Star Inc., Ashland,
OR). THP-1 staining was performed with antibodies to CD14,
CD83, HLA-DR from Biolegend (San Diego, CA) and CD80/
CD86 and CCR7 from BD Bioscience (San Jose, CA).
Cytotoxicity assay
The cytolytic activity against target cells was assayed using
previously characterized HTLV-1 Tax11-19 (LLFGYPVYV)-
specific CD8+ CTL clone [29]. The CTL clone was maintained
by weekly stimulation with peptide-pulsed (1 mg/ml) irradiated
PBMCs from an HLA-A201+ non-HTLV-1 infected individual.
CTL culture medium was IMDM containing 10% human serum,
2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml strepto-
mycin. Human recombinant interleukin-2 (50 U/ml) was added
on the next day of stimulation. Target cells were CD4+ T-cells
infected with HTLV-1 D26, N26, G29S or 12KO and autologous
Epstein-Barr virus-transformed B-cells as a positive control. The
cytotoxicity assay was performed using DELFIA EuTDA Cyto-
toxicity assay (Perkin Elmer). Target T-cells were loaded with bis
(acetoxymethyl) 2,29:69,20-terpyridine-6,60-dicarboxylase (BATD
A) and pulsed with or without 100 ng/ml of Tax peptide. Target
cells (36103) were incubated with CTL clones for 3 hours at 37uC
in 96 well plates at indicated effector-to-target ratios. The
supernatant (20 ml) was incubated with 200 ml of Europium
solution, and the fluorescence was measured in a fluorometer
(Wallac 1420 VICTOR3; Perkin Elmer). The percent specific lysis
was calculated as (experimental release2spontaneous release)/
(maximum release2spontaneous release)6100. The assay was
performed in triplicate.
Supporting Information
Table S1 Extracellular p19Gag production and viral DNA copy
in HTLV-1 producer B-cells.
(DOCX)
Table S2 Characterization of HTLV-1 infected CD4+ T-cells.
(DOCX)
Table S3 Viral DNA loads for patients with G29S mutations.
(DOCX)
Text S1 Genbank accession numbers for orf-I sequences from
HTLV-1 infected individuals.
(TXT)
Acknowledgments
We thank Teresa Habina for editorial help, T. Misteli for critical reading of
the manuscript, H.K. Chung, G. Ferrari, S. Orndorff, and J. Treece of
Advanced BioScience Laboratories, Inc. for help with the macaque studies
and V. Ciminale and F. Rende for helpful discussions.
Author Contributions
Conceived and designed the experiments: CAPM GF. Performed the
experiments: CAPM MFdCA YEA RCB RWP CF DE MF LCJA IB JG.
Analyzed the data: CAPM KM DV GF JCW. Contributed reagents/
materials/analysis tools: BGC AG SJ. Wrote the paper: CAPM GF.
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