Human T-Lymphotropic Virus (HTLV) Type I in vivo Integration in Oral Keratinocytes

Abstract

Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (HAM/TSP) patients and 11 asymptomatic carrier individuals (AC) coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14% (8/14) HAM/TSP patients and 27.28% (3/11) AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.

Keywords: Human T-Lymphotropic virus type 1; Oral keratinocytes; Oral mucosa; Proviral integration; sIgA.

Figure 1.

Schematic localization along the HTLV-1 proviral genome of several oligonucleotide primers pairs that were used to amplify HTLV-1 proviral sequences from DNA extracted of infected oral keratinocytes. Direction of arrows show the sense of each one of HTLV-1 oligonucleotide primers. Supplementary information about the primers is described in material and methods.

Figure 2.

Reactivity detected by western blot in plasma and OF against HTLV-1 proteins of one HAM/TSP patient from southwest Colombia. (A). Western blot using the commercial HTLV blot 2.4 assay kit (Genelabs^® Diagnostics Pte Ltd. Singapore Science Park. Singapore). (1) plasma control for HTLV-1 included with the kit; (2) plasma control for HTLV-2 included with the kit (3) Anti-IgG HTLV-1 in plasma; (4) Anti-IgG HTLV-1 in OF; (5) Anti-sIgA in OF; (6) Negative plasma control. (B). Western blot with a Tax recombinant protein of a HAM/TSP patient. (1) anti-r-tax -IgG in plasma. (2) anti-r-tax -IgG in OF. (3) anti-r-tax-sIgA in OF (4) Negative OF control. (C). Frequencies of sIgA HTLV-1 specific class reactivity of HAM/TSP patients and AC OF from southwest Colombia against the different HTLV-1 proteins detected by western blot. (HAM/TSP). Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy. (AC). Asymptomatic Carrier.

Figure 3.

Two color flow citometry analyses of pairs of CD antigens as cell membranes markers in primary cultures from oral mucosa cells and keratin detection with the polyclonal antiserum A-575 (Daco, Carpinteria, CA) using the Vectastain Elite ABC kit (Vector, Burlingame. CA) kit. (A to C). FACS analyses in oral mucosa cells after 24 hours of cultured. (D to F). FACS results in oral mucosa cells after five days of cultured. (G) Immune staining of oral mucosa cells cultures after five days with the polyclonal antiserum A-575 (40X). (H). Negative Control IgG (40X).

Figure 4.

Southern blot hybridization of PCR products obtained from DNA of five days old primary oral keratinocytes from HAM/TSP and AC using different pairs of HTLV-1 oligonucleotide primers. LTR (737bp); Pol (189 bp); Pol-Env (1033 bp); Tax (159 bp). (MT) DNA from MT2 cell line with HTLV-1 provirus. Appropriated ^[32]-P-labeled oligonucleotides were used as probes.

Figure 5.

Southern blot hybridization from RT-PCR products of (A) Tax and (B) Pol polyA⁺ RNA obtained from oral epithelial cells of those HAM/TSP patients and Healthy Carriers. who had detectable levels of sIgA in their oral fluids. (C) An internal β-actin gene reporter 159 bp fragment was included as a control for transcription of cellular genes. Appropriated ^[32]-P-labeled oligonucleotides were used as probes. (1 a 12), RT-PCR products of polyA+ RNA extracted from HAM/TSP patients; (13 a 15), RT-PCR products of RNA from asymptomatic carriers (AC); (16) RT-PCR of Poly A+ RNA from Peripheral Blood Mononuclear Cell (PBMC) of a HAM/TSP patient; (17). RT-PCR of poliA+ RNA from from MT2 cell line with HTLV-1 provirus. (18) PCR of MT2 DNA cell line with HTLV-1 provirus.