First report of HTLV-1 truncated p12 protein in Brazil

KEYWORDS:

• HTLV-1
• mutation
• p12

HTLV-1 infects approximately 5–10 millions of individuals in the world and the majority of them remain lifelong asymptomatic carriers (AC). However, up to 5% of HTLV-1 patients may present inflammatory neurological or proliferative disorders as HTLV-1- associated myelopathy / tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL), respectively. In HAM/TSP, the spinal cord inflammation, leads to a chronic disabling disturbance. In ATL, HTLV induces T-cell proliferation and transformation, with a lethal consequence to the host.¹ High proviral load is associated with disease progression. However, factors that may influence viral replication are unclear. Some viral encoded proteins, such as p12, induces infected T-cell proliferation and stimulates immune evasion.^2-15 Previous studies demonstrated that mutations in p12 may influence the HTLV-1 infection outcome.^3,5,8,16,17 Here, we identified, at the first time, a HTLV-1 strain with a premature stop codon in p12 infecting an individual from Brazil, presenting a very low proviral load. We hypothesize if this mutation would have a protective role against HTLV-1 replication.

Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) of an asymptomatic individual that is followed in the Neuroinfection outpatient clinic (HUGG/UNIRIO). The patient was a Brazilian Caucasian, 53 years old, female, married, with no family history of HTLV-1 infection, blood transfusion or drug abuse. The HTLV-1 infection was detected by ELISA and Western Blot fifteen years ago, when the patient was tested for blood donation. The HTLV-1 proviral load was 0.03 copies of Tax/100 PBMCs (TaqMan Real Time PCR).¹⁸ For DNA extraction QIAamp DNA mini kit (Quiagen, CA) was used. Nested polymerase chain reaction (n-PCR) was performed in order to amplify p12¹⁸ and LTR. The PCR product was purified using Illustra GFX PCR DNA and Gel band purification kit (GE HealthCare) and directly sequenced using ABI model 373A automated DNA sequencer and the manufacturer's Dye Terminator FS Kit instructions. The electropherogram was analyzed by Chromas Lite 2.1 (Thechnelysium) (Fig. 1). The p12 sequence was aligned and translated on BioEdit editor (North Carolina State University). The analyzed sequence (HUGGRJ25) presented a mutation (Fig. 1) that resulted in a premature stop codon at position 82 (Genbank accession number: KR337634) (Fig. 2). The phylogeny on LTR (634bp) showed that the sequence belongs to HTLV-1aA subtype (GenBank accession number KR337613).

Figure 1. Electropherogram of DNA sequence obtained from peripheral blood of a Brazilian asymptomatic individual. The point mutation (G245A) that resulted in the premature stop codon is highlighted and indicated with an arrow. It is localized at position 286 in the present electropherogram.

Figure 2. HTLV-1 p12 alignment. Predict aminoacid sequence of p12 of HTLV-1 asymptomatic carrier from Rio de Janeiro, Brazil (HUGG25), HTLV-1aA Prototype (BOI: L36905) and other previously described p12 truncated sequences that are available on GenBank.*: represents stop codon; HAM: HAM/TSP individuals; AC: HTLV-1 asymptomatic carriers; WA: West Africa; CA: Central Africa. The cleavage sites (yellow) and the ubiquitylation (88) positions (red) are highlighted. SH3-binding motifs are underlined. Calcineurin-binding motif is shown in bracket.

Truncated p12 proteins had already been identified in AC, HAM/TSP and ATL individuals from Argentina, Japan as also as in Simian T Lymphotropic virus (STLV).¹⁹ In this set of sequences two truncated p12 proteins were observed: the major group presented 87 residues and the other 81 residues. Our truncated p12 sequence belongs to the later type. One patient from Japan with a truncated protein (with the stop codon at position 82) was a HAM/TSP and presented a high proviral load.²⁰ This is the first report of a truncated protein in a HTLV-1aA from Brazil. In fact, we evaluated a large set of p12 sequences available in GenBank from 76 new HTLV-1 complete genome sequences from Brazil²¹ and none presented a premature stop codon at p12 protein. Moreover, Igñez et al (2005) analyzed 26 ORF-I sequences from Brazil (20 from AC, and 6 from HAM/TSP) and also did not find any individual carrying a provirus with a truncated p12.²² In a very interesting study, ORF-I sequences of 160 HTLV-1 infected individuals from different areas of the world (including Brazil) were cloned and none of 1600 obtained sequences had a premature stop codon either.³ In Japan, the prevalence of truncated p12 protein was 4% (n = 10) among HTLV-1 infected individuals.²⁰

An in vitro study demonstrated that strains producing a truncated p12 protein (without the amino acids 87–99) presented a dramatic reduction of NFAT activation.²³ As NFAT activation is associated with the upregulation of IL-2, this deletion may cause a decrease on T-cell proliferation.²³ The p12 protein presents different mechanisms to induce T-cell activation and proliferation. One mechanism is associated with NFAT activation. It is know that p12 localizes at the endoplasmatic reticulum (ER), where it can interacts with different cellular proteins. It can bind to calreticulin/calnexin, increasing the release of calcium from ER. The increase in the cytosolic calcium, activate the calmodulin, which stimulates calcineurin (calmodulin phosphatase dependent). Calcineurin dephosphorilates NFAT, activating it. Then, NFAT is translocated into the cell nucleus where it increases the IL-2 transcription.⁶ In the other hand, p12 can bind to calcineurin at the ER, competing with NFAT. This induces a negative modulation of NFAT activation.^6,24 It is know that there is a calcineurin/calnexin binding motif in p12 protein, localized in position 70–75.²⁴ It was also demonstrated that there are four different SH3 binding motifs in p12. While SH3–2 and SH3–4 presents a positive regulation on NFAT, SH3–1 and SH3–3 presents a negative impact of NFAT activation.²³ Therefore, NFAT regulation by p12 is complex. The truncated form still present the calcineurin binding motif, two inhibitory SH3 motifs and only one SH3 with a positive regulation of NFAT. The same picture was observed in the truncated protein produced by Ding et al (2003), which presented a marked reduced NFAT activation in vitro.²³

Moreover, the p12 protein undergoes to complex post-translational modifications. It can be cleaved at two positions (at position 9 and 29). When p12 is cleaved at position 9 it lost the ER retention signal. When it is cleaved at position 29 it produces the p8 protein. While p12 remains at the ER, p8 traffic to cell surface where it plays an important role. At the ER, p12 can bind to different molecules: 1) IL-2R (increasing the Jack/STAT signaling), 2) MHC-I heavy chain (impairing the expression of this molecule), 3) Calnexin/calmodulin (increasing cytosolic calcium, activating NFAT). In contrast; p8 localizes at immunological synapses (IS) and interacts with TCR, causing a down modulation to the TCR signaling. Moreover, p8 increases cell adhesion and viral transmission via cell-cell synapses. These mechanisms are important to maintain a persistent viral infection, as it interferes in both immune response and viral transmission. The exact cellular pathway that p8 utilize for achieve this objectives are not elucidated. By hypothesis, the C-terminus truncated protein can influence in both p12 and p8 function.

Others post translational modifications identified in p12 includes ubiquitylation, palmitoylation and dimerization.^25-28 Some mutations, such as C39A abrogated dimerization and palmitoylation of both proteins, without interfering neither in the localization of p8 nor in the increase in the cellular adhesion. As C39 is maintained in our truncated p12, the palmitoylation may occur in this protein.^27,28 In vitro studies showed that ubiquitylation occur at a Lysine localized at position 88, and it influences the protein stability.²⁵ p12 is ubiquitylated and subsequently degraded in the proteasomes. For example, a mutation to an Arginine in position 88 (K88R), inhibits the ubiquitylation and increases p12 stability.²⁵ Therefore, as the premature stop codon is on position 82, it may influence the protein stability.

The protein localization is important for it function. In this context, a mutant lacking the C-terminal amino acid residues (1–86) maintained the wild type p12 protein localization.⁷

Different p12 alleles present different profiles of p12 and p8 expression. Interestingly, all described truncated proteins presented alleles that were previously associated with an increased expression of p12 (F3L, S23P, F34L/F61L, S63P).³ A transfection study, as performed by Pise-Masison et al. (2014) to evaluate the expression profile of both proteins would be interesting.

It is possible that, in situations with limited concentration of IL-2 (as usually occurs in vivo) p12 will be important in the initial phase of infection (causing a T cell activation and proliferation and immune evasion).²⁹ At a later stage, p8 becomes more important causing infected T cell anergy and increasing cell-cell adhesion to transfer viral particles through the virological synapses. The post-translational mechanisms could control these opposing effects by changing the production profile of p8 and p12, according to the stage of infection (mechanism similar to what occurs with HIV Nef). Finally, in situations where the individual presents high levels of IL-2, the importance of p12 for HTLV-1 replication would be limited. This may occur in the presence of some co-infections, such as with Hepatitis C virus (HCV).³⁰ This hypothesis may explain the differences observed between this current case and previous described patients.²⁰

This is the first report of a truncated p12 protein in a patient from Brazil presenting a very low proviral load.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The PDTIS/FIOCRUZ sequencing platform.

Funding

This work was supported by Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and by Post-Graduation on Infectious and Parasitic Diseases (UFRJ), a PhD fellowship to C.R. from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and a PhD fellowship to M.J.C.C. and L.Z. from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).