Role of the HTLV-1A and HTLV1-C inflammatory profile in disease

Division Of Basic Sciences - Nci

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Abstract

Human T-cell leukemia virus type I (HTLV-1) causes the neurodegenerative disease Tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM) and inflammatory conditions such as dermatitis, arthritis, and uveitis. HTLV-1 infection is endemic in regions worldwide and despite the high genetic conservation of HTLVs, seven global subtypes have been identified based on the sequences of their viral promoters, long terminal repeats (LTRs), and envelopes. HTLV-1A is the most common type globally. HTLV-1C, the most divergent variant, is endemic among Aboriginal populations in the Northern Territory, Australia, with a seroprevalence above 30%. Clinical data from this population suggest a higher association of HTLV-1C infection with lung morbidity, possibly stemming from a genetic difference in the HTLV-1 subtypes. The highest nucleotide divergence in the genomes of HTLV-1 A and C occurs in orf-I, encoded by a singly spliced mRNA in HTLV-1A, but lacking a translation initiation codon in HTLV-1C. Prior work has demonstrated that orf-I expresses p12 and p8; proteins central to HTLV-1A persistence that increase STAT-5 signaling, downregulate the expression of MHC-I, ICAM-1, and ICAM-2, and bind to the 16 kDa subunit of vacuolar ATPase. The ER retention signal of p12 is cleaved to generate p8, which localizes to the cell surface and downregulates TCR signaling, induces conduit formation, and rapidly transmits virus to neighboring cells. Indeed, p8 binds to vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation and facilitates Gag transfer via cellular conduits. Genetic mutations at the p12 cleavage site affect the balance of p12/p8, which itself is associated with virus levels in infected individuals. Both p12 and p8 are essential for viral infectivity, as ablation of orf-I expression or genetic mutations affecting the balance of p12/p8 impairs HTLV-1 infectivity in the macaque model. While orf-I expression is central to HTLV-1A infectivity, early findings that HTLV-1C lacks the AUG initiation codon for orf-I translation have recently been corroborated, leading us to hypothesize that HTLV-1C expresses orf-I by an alternative mechanism. To test our hypothesis, and since export of primary blood samples from Aboriginal people living with HTLV-1C to the United States is restricted, we constructed a chimeric HTLV-1A/CoI-L molecular clone in silico and demonstrated its infectivity in human and macaque CD4+ T-cells in vitro and in macaques in vivo. We found that subtype C orf-I (orf-IC) can be expressed in vitro and in vivo in the lungs of infected macaques by a doubly spliced mRNA juxtaposed with the first exon of rex, which provides its ATG in frame with orf-I and encodes the p16C protein (rex-orf-IC). Although comparison of HTLV-1A and chimeric HTLV-1A/CoI-L viruses demonstrated similar systemic infectivity in macaques, the host inflammatory response in blood and bronchoalveolar lavage (BAL) differed significantly. Importantly, animals infected with HTLV-1A/CoI-L had higher levels of viral expression in the lung than those infected with HTLV-1A and developed interstitial pneumonia with infiltration by T-cells and B-cells, peripheral fibrosis, and bronchiectasis. Minimal lung viral expression and minimal lung pathology were observed in HTLV-1A infected animals. In vitro, T-cells expressing p16C become resistant to engulfment by efferocytosis, a monocyte function that clears apoptotic cells and maintains tissue homeostasis. These data suggest the hypothesis that p16C expression in the lung of HTLV-1A/C infected animals may mediate the pathogenetic mechanism underlying the increased inflammation and lung disease, as efferocytosis is essential for lung homeostasis. Additionally, we developed a high-throughput flow cytometry-based assay system to measure HTLV-1 envelope-specific ADCC. We used a natural killer cell-resistant T-lymphoblastoid cell line stably expressing the green fluorescent protein GFP to construct a target cell line expressing HTLV-1 envelope protein and using monoclonal antibodies and plasma samples from HTLV-infected or uninfected individuals, validating the specificity and sensitivity of the assay.