Characterization and sequencing of prototypic human T-lymphotropic virus type 1 (HTLV-1) from an HTLV-1/2 seroindeterminate patient

Abstract

Serological screening for human T-lymphotropic virus type 1 (HTLV-1) parallels the standard screening process for human immunodeficiency virus (HIV), in which samples found positive by enzyme-linked immunosorbent assay (ELISA) are confirmed with a modified Western blot procedure. There are a significant number of cases in which HTLV-1/2 ELISA-positive specimens demonstrate an incomplete banding pattern on this Western blot. Individuals providing these atypical antibody responses are categorized as seroindeterminate for HTLV-1/2. Although HTLV-1 genomic sequences are readily detectable in the peripheral blood lymphocytes (PBL) of seropositive individuals, previous studies have repeatedly demonstrated that PBL from the vast majority of HTLV-1/2 seroindeterminate individuals are PCR negative for HTLV-1. As a result, identification of the agent responsible for this indeterminate reactivity has been of interest. We have generated an HTLV-1-positive B-cell line (SI-1 B) from one of these seroindeterminate individuals. Previous screening for HTLV-1 in PBL from this patient had been routinely negative by primary PCR; however, HTLV-1 tax had been periodically detected by nested PCR. DNA sequence data generated with genomic DNA from the SI-1 B cell line and HTLV-1-specific primers demonstrated the presence of a full-length viral genome with >97% homology to the Cosmopolitan form of HTLV-1. A 12-bp deletion was identified in the 3'-gag/5'-prot region, which would predict translation of altered or nonfunctional proteins from these genes. We propose that this HTLV-1/2-seroindeterminate patient is infected with a prototypic form of HTLV-1 at an extremely low viral load and that this finding may explain HTLV-1/2 seroindeterminate reactivity in at least a subset of these individuals.

FIG. 1

HTLV-1/2 Western blot. Serum from a representative HAM/TSP patient was blotted from 1:50 to 1:160,000 dilutions. Serum from seroindeterminate patient SI-1 was blotted from 1:50 to 1:10,000 dilutions. Reactivity to the p24 antigen and to either rgp21 or rgp46 (∗) is required for a classification of HTLV-1 seropositivity.

FIG. 2

PCR for HTLV-1 tax using genomic DNA isolated from an HTLV-1-infected HAM/TSP B-cell line and the SI-1 B cell line.

FIG. 3

Southern blot for HTLV-1 integrated in B-cell genomic DNA generated from a patient with HAM/TSP (lanes 1 and 2), the SI-1 B cell line (lanes 3), and an HTLV-1-infected cell line from a patient with ATL (lanes 4). The line from the ATL patient contains a monoclonal integration of the HTLV-1 provirus. The other lines harbor multiple integration sites of the virus. Following digestion with PstI, expected virus-specific bands are 2.4, 1.6, and 1.3 kb; additional bands at higher molecular sizes are attributed to viral sequences contiguous with cellular genomic DNA external to adjacent PstI sites in the viral genome. Digestion of prototype virus with EcoRI predictably yields bands greater than 9 kb, due to a lack of internal EcoRI sites.

FIG. 4

(a) Antigen capture assay for HTLV-I Gag. Hut102 and MT-1 are HTLV-1-infected T-cell lines; MT-1 is known to have very low levels of viral transcriptional activity. UN-1 is a B-cell line generated from an HTLV-1-seronegative individual. Black and grey columns represent cell supernatant and cell lysate fractions, respectively. (b) Western blot assay for HTLV-1 p19. Western blot analysis was performed on supernatants (Spt) and cell lysates (Ly) from an uninfected B-cell line (UN-1), a B-cell line derived from seroindeterminate patient SI-1 (SI-1 B), a B-cell line from a HAM/TSP patient (HAM/TSP-1), and an HTLV-1 infected T-cell line (Hut102) with a monoclonal antibody to HTLV-1 p19 at a 1:50 dilution. SeeBlue (Novex, San Diego, Calif.) markers (not shown) were used for gel calibration. (c) RT-PCR assay for HTLV-1 tax, pol, and env. UN-1 is an HTLV-1-uninfected B-cell line; Hut102 is an HTLV-1-infected T-cell line; Jurkat is an HTLV-1-uninfected T-cell line; HAM/TSP is an HTLV-1-infected B-cell line from a HAM/TSP patient: SI-1 is a B-cell line derived from seroindeterminate patient SI-1.

FIG. 5

(a) Schematic representation of the HTLV-1 genome and relative locations of overlapping PCR clones generated from the SI-1 B cell line. (b) Overlapping PCR clones generated from the SI-1 B cell line using primers specific for prototypic HTLV-1. Primer sequences and expected amplimer sizes are shown in Table 1.