# Truncated MTA-1: A Pitfall in ELISA-Based Immunoassay of HTLV-1 Infection

Authors: Mohammad Reza Abbaszadegan; Narges Jafarzadeh; Mojtaba Sankian; AbdolReza Varasteh; Mahmoud Mahmoudi; Majid Sadeghizadeh; Fatemeh Khatami; Neema Mehramiz
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

HTLV-1 causes adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Recombinant envelope glycoprotein is used in production of diagnostic enzyme-linked immunosorbent assay (ELISA) kit. There are some reports that a significant percentage of Iranian HTLV-1 infected patients showed no seroreactivity with MTA-1 peptide, while HTLV-1 had been confirmed by PCR detection methods or ELISA kits containing a cocktail of HTLV-1 specific peptides. This report describes experiments designed to determine whether some discrepancies between ELISA and PCR results could be due to truncation of immunodominant epitopes using immunoassay method. We have cloned the MTA-1 epitope of env gene from HTLV-1  in NotI/NdeI sites of pET22b(+) expression vector. Sequencing analysis of recombinant plasmids revealed an insertion of a cytosine in position 271 causing a stop codon in the MTA-1 protein translation. SDS-PAGE analysis also failed to reveal the presence of the desired protein. Subjects with a mutant HTLV-1 env gene were shown to be seronegative using ELISA, but positive with PCR.


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## Body

## 1. Introduction

Infection with human T-cell lymphotropic virus type-1 (HTLV-1)
is a growing medical problem, with over 20 million estimated infections
worldwide [1].  HTLV-1 has been identified as the etiologic agent of two
distinct human diseases:  adult T-cell leukemia (ATL) and a chronic, progressive
demyelinating disorder known as HTLV-1-associated myelopathy/tropical spastic
paraparesis (HAM/TSP) [2]. The virus has also been associated with a number of
autoimmune diseases, including Sjögren’s syndrome, uveitis, and an
inflammatory arthropathy [3]. The regions of HTLV-1 endemicity, with
proportionately higher rates of infection, are clustered in southern Japan, the Caribbean, South America, the
southern United States,
equatorial Africa, and northeastern Iran [4, 5]. Three major routes of
HTLV-1 transmission are mother to child via breast milk, sexual intercourse,
and blood transfusion [6, 7]. Otherwise, HTLV-1 is not easily transmissible,
since cell-to-cell contact is presumably required [6, 8]. Retroviruses can
employ a variety of different cell surface proteins as receptors for binding
and entry into host cells. Entry involves an interaction between the surface
protein (SU) of the virus envelope glycoprotein (env) and the cellular receptor
with subsequent fusion of the cellular and viral membranes mediated by the
transmembrane (TM) region of env [9, 10]. According to current
diagnostic criteria, immunoreactivity with p24 and one of the envelope proteins
(usually gp46) is considered diagnostic of HTLV-I or HTLV-II infection;
however, this immunoreactivity pattern still results in a significant
proportion of false negative. MTA-1 peptide from the HTLV-1 gp46 region demonstrated
the highest percentage of reactivity with HTLV-I positive sera, particularly
among subjects of Japanese ancestry [11, 12]. There are some reports that a
higher percentage of Iranian HTLV-1 infected patients showed no seroreactivity
with MTA-1 peptide, while HTLV-1 infection of patients had been confirmed by
PCR detection methods or ELISA kits containing a cocktail of HTLV-1 specific
peptides [4].Some of these
discrepancies could be explained by variation in immune response at different
population. The other cases may be as a result of truncation of gp46 region in
HTLV subtybes. Previously, it has been demonstrated that point mutations or
deletion of glycosylation sites can result in a nonfunctional env protein [13].This report describes
experiments designed to determine whether some discrepancies between ELISA and
PCR results could be due to truncation of immunodominant epitopes using
immunoassay method.


## 2. Materials and Methods

To prepare MTA-1 insert DNA, primers were chosen based
on their locations within the prototype HTLV-1 genomic sequence available in
the Gen-Bank database with accession number AF.33817. In order to put sticky end into blunt-ended
amplified molecule, the sequences of restriction enzymesNde I and Not
I were incorporated into the 5′ ends of forward and reverse primers,
respectively, with regard to cloning site of pET-22b(+) vector (Novagen,
Madison, WI, USA), and restriction sites flanked by 3 spacer nucleotides at the
5′ end to allow for efficient digestion. The PCR amplification of DNA fragment
of interest was performed for 35 cycles at the following temperatures: 10
cycles; 1 minute at 94°C, 45 seconds at 55°C, 2 minutes at 72°C, and 25 cycles;
45 seconds at 94°C, 1 minute at 65°C and 2 minutes at 72°C with a final
extension of 20 minutes at 72°C in a DNA thermal cycler (Perkin-Elmer 480). Final
composition of the reaction was 100 ng of pool of four DNA samples, 20 pmole appropriate primers, 1.5 mM dNTP mixture, and one unit (u) of Pfu DNA polymerase in the buffer
containing MgSO4 in 25 μl
reaction volume. Sequences of the primers were as follows.Forward primer:5′-GCGCATATGGCTCCAGGATATGACCCCATC-3′.Reverse primer:5′-ATAGCGGCCGCGGAGCGGGATCCTAGGGTG-3′.Expected product size of 455 bp fragment was amplified,
gel purified using CoreOne gel extraction kit. Amplified DNA insert was
sequentially digested and followed by a second gel purification.Double digestion of plasmid DNA (5493 bp) was performed
usingNot I and Nde I restriction enzymes at 37°C, overnight.
After purification, cut plasmid DNA was dephosphorylated with calf intestine
phosphatase enzyme. The modified and digested pET-22b(+) plasmid was gel purified prior to ligation with the insert.The appropriate amount of insert used in the ligation
assay was calculated using the following equation: [(amount of vector, ng) × (size of insert, kb)/(size
of vector, kb) × (molar
ratio of insert/vector)] = ng of insert DNA. pET-22b(+)/MTA-1 constructed by
ligating MTA-1 sequence into the pET-22b(+) expression vector viaNot I–Nde I sites, and the reactions were
carried out by T4 DNA ligase enzyme in 10 μl volume. T4 DNA ligase was then
inactivated by heat treatment for 20 minutes at 65°C to increase the efficiency
of transformation. Plasmid carrying the gene was used to transform bacterial
cells (E. coli DH5a) using Inoue transformation method.The cells were grown overnight at 37°C on SOB plates
containing 50 μg/mL ampicillin. Plasmid DNA was extracted using Bioneer plasmid
extraction kit (Bioneer, Korea, USA); screening was performed using
PCR colony with insert-specific PCR primers, and restriction analysis and
recombinant plasmid sequenced.For protein analysis, a recombinant pET-22b(+)/MTA-1 plasmid
was transformed into expression hostE. coli BL21(DE3) strain containing
a chromosomal copy of the gene for T7 RNA polymerase. Preparation of
recombinant cells was followed by induction and optimization of expression of target
protein using a range of IPTG concentration from 0.1 mM to 1 mM added to a
growing culture. Cells were collected from the growth medium by centrifugation,
14000-rpm, 1 minute, at room temperature. The pelleted cells were resuspended
in extraction buffer (50 mM potassium phosphate, pH 7.8, 400 mM NaCl, 10%
Glycerol, 100 mM KCl, and 0.5% Triton X-100) and subjected to centrifugation at
14,000 rpm for 20 minutes. Supernatants were removed; 250 μL 2X SDS/PAGE sample
buffer was added to cells and equal volumes of supernatant mixed with 2X sample
buffer, incubated at 95°C for 5 minutes and subjected to reducing sodium
dodecyl sulfate SDS-polyacrylamide gel electrophoresis (PAGE).


## 3. Results and Discussion

pET-22b(+) vectors were chosen as a convenient and powerful
system for cloning and expression of recombinant protein inE. coli [14]. Extraction of plasmid, digestion, isolation, ligation, transformation,
identification, and PCR were performed as described previously [15]. The
conditions of the ligation reactions were first subjected to a number of
optimizations for various parameters [16]. The temperature of 16°C resulted in
desirable result. In the experimental sets focusing on the amount of T4 DNA
ligase, both 10 and 20 units of enzyme gave high efficiency. The time of the
ligase reaction was influential as 4-hour ligation reaction resulted in a high
number of recombinant plasmids than the overnight ligation.One of the strategies for high-efficiency cloning of
sticky-end DNA molecules involves dephosphorylation of vectors with calf
intestinal phosphatase (CIP), which catalyses the hydrolysis of5′-phosphate
groups from DNA. Treatment with CIP also increased the ligation efficiency of
the PCR-amplified inserts.Using these strategies as well as the optimized ligation
conditions, three different molar ratios of insert to vector DNAs were tested.
The ratios of1:1 gave significantly low transformation efficiency, while the
insert to vector ratio of 3:1 resulted in the highest number of transformants.As shown in Figure1, Lanes 2 and 4 showed digested plasmid from
transformed clones with no insert using Alw 441 restriction enzyme. One
of three transformed clones (Lane 3) contained recombinant pET-22b(+) carrying
a 455 bp fragment. The presence of this fragment changed Alw 441 restriction map of
recombinant pET-22b(+) (Figure 1, Lane 3) in comparison with nonrecombinant
pET-22b(+) (Figure 1, Lane 5).Figure 1
Alw 441 restriction analysis
of plasmids, extracted from transformed clones. Plasmids of transformed clones are visualized on ethidium
bromide-stained 1% agarose gel after purification and digestion with Alw 441. Lane 1:1 kb DNA Ladder; Lane 2, 4: digested plasmid from transformed clones with
no insert using Alw 441 restriction enzyme; Lane 3, a transformed clone containing
pET-22b(+) with a 455 bp MTA-1 insert; Lane 5 indicates Alw 441 restriction
map of nonrecombinant pET-22b(+) (negative control).Sequencing analysis showed an insertion of a cytosine
in position 271 causing a frame shift and stop codon in protein translation
(Gen-Bank accession no. DQ911752) (Figure2). All four DNA samples used in this
study send for sequencing and showed the same results. For further investigation,
SDS-PAGE analysis was performed to detect potential MTA-1 protein. However, this
analysis failed to reveal the presence of the desired protein band 37 KDa. It
is likely that the MTA-1 truncated protein resulted from 271insC mutation is an
unstable and degradable protein giving no band in SDS-PAGE.Figure 2
Sequencing analysis. An insertion of a
cytosine in position 271 causing a stop codon in the MTA-1 protein translation.Screening for HTLV-1 has become routine in blood banks
in the most developed nations. The screening process is initiated with an
HTLV-1-specific enzyme-linked immunosorbent assay (ELISA).  Samples which are
found to be repeatedly positive by ELISA are confirmed through a PCR assay or,
more commonly, a commercially available Western blot assay. The standardized ELISA
assay incorporates viral proteins obtained from a number of recombinant HTLV-1 glycoproteins,
which are included to increase the sensitivity and specificity of the assay. In
a significant number of cases, which can be demonstrated throughout the world,
the HTLV-1/2 ELISA is negative, but
HTLV-1 PCR is positive. The causative agent(s) and medical significance of the HTLV-1
seroindeterminate status are unclear. A number of potential explanations have
been provided for this serological finding, including (i) infection with a truncated
or deleted form of HTLV-1, (ii) infection with a novel retrovirus bearing
significant homology to HTLV-1, and (iii) infection with prototype HTLV-1 at
viral loads which are below the range of current methods of detection. Previous
studies have had limited success in addressing these possibilities.The presence of truncated form of HTLV-1 gp46 protein
could have been implicated as potential agents of disease in a number of cases.
Such truncated form of HTLV-1 could theoretically account for an incomplete
banding pattern on an HTLV-1-Western blotting due to absence or alteration of crucial immunodominant viral epitope.The 271insC mutation in
MTA-1 envelope genomic sequences causing a truncated protein can explain the
seronegative (ELISA negative) HTLV-1 infected individuals who are HTLV-1 PCR positive.
Although samples found to be repeatedly negative by HTLV-1-ELISA, HTLV-1 genomic
sequences were readily detectable by PCR in the peripheral blood lymphocytes of these infected
individuals (Figure3). The positive results were also confirmed by sequencing.Figure 3
Amplification of HTLV-1 MTA1-coding region. A 455 bp fragment
was amplified from the MTA-1 region of HTLV-1 genome. PCR products were electrophoresed
in a 1.5% agarose gel, stained in ethidium bromide, and photographed. Lane 1,
100 bp ladder; lane 2, negative control; lane 3, PCR product from an ELISA
positive patient; Lane 4, PCR product from an ELISA negative patient.Therefore, in addition to
variation in immune response at different population, some of the discrepancies
between PCR detection methods and ELISA kits containing MTA-1 peptide from the
HTLV-1 gp46 region could be explained by truncation of gp46 region in HTLV
subtybes. In consistent with this notion, it has been demonstrated that point
mutations or deletion of glycosylation sites can result in a nonfunctional env protein [13].



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*Source: 846371-2008-06-11.xml*