Maria Fernanda de Castro-Amarante

Orf-I and Orf-II-Encoded Proteins in HTLV-1 Infection and Persistence

The 3′ end of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. Here, we review current knowledge of HTLV-1 orf-I and orf-II protein products. Singly spliced mRNA from orf-I encodes p12, which can be proteolytically cleaved to generate p8, while differential splicing of mRNA from orf-II results in production of p13 and p30. These proteins have been demonstrated to modulate transcription, apoptosis, host cell activation and proliferation, virus infectivity and transmission, and host immune responses. Though these proteins are not essential for virus replication in vitro, p8, p12, p13, and p30 have an important role in the establishment and maintenance of HTLV-1 infection in vivo.

Co-dependence of HTLV-1 p12 and p8 Functions in Virus Persistence

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.

Human T Cell Leukemia Virus Type 1 Infection of the Three Monocyte Subsets Contributes to Viral Burden in Humans

ABSTRACT Because the viral DNA burden correlates with disease development, we investigated the contribution of monocyte subsets (classical, intermediate, and nonclassical monocytes) to the total viral burden in 22 human T cell leukemia virus type 1 (HTLV-1)-infected individuals by assessing their infectivity status, frequency, as well as chemotactic and phagocytic functions. All three monocyte subsets sorted from HTLV-1-infected individuals were positive for viral DNA, and the frequency of classical monocytes was lower in the blood of HTLV-1-infected individuals than in that of uninfected individuals, while the expression levels of the chemokine receptors CCR5, CXCR3, and CX3CR1 in classical monocytes were higher in HTLV-1-infected individuals than uninfected individuals; the percentage of intermediate monocytes and their levels of chemokine receptor expression did not differ between HTLV-1-infected and uninfected individuals. However, the capacity of intermediate monocytes to migrate to CCL5, the ligand for CCR5, was higher, and a higher proportion of nonclassical monocytes expressed CCR1, CXCR3, and CX3CR1. The level of viral DNA in the monocyte subsets correlated with the capacity to migrate to CCL2, CCL5, and CX3CL1 for classical monocytes, with lower levels of phagocytosis for intermediate monocytes, and with the level of viral DNA in CD8+ and CD4+ T cells for nonclassical monocytes. These data suggest a model whereby HTLV-1 infection augments the number of classical monocytes that migrate to tissues and become infected and the number of infected nonclassical monocytes that transmit virus to CD4+ and CD8+ T cells. These results, together with prior findings in a macaque model of HTLV-1 infection, support the notion that infection of monocytes by HTLV-1 is likely a requisite for viral persistence in humans. IMPORTANCE Monocytes have been implicated in immune regulation and disease progression in patients with HTLV-1-associated inflammatory diseases. We detected HTLV-1 DNA in all three monocyte subsets and found that infection impacts surface receptor expression, migratory function, and subset frequency. The frequency of nonclassical patrolling monocytes is increased in HTLV-1-infected individuals, and they have increased expression of CCR1, CXCR3, and CX3CR1. The viral DNA level in nonclassical monocytes correlated with the viral DNA level in CD4+ and CD8+ T cells. Altogether, these data suggest an increased recruitment of classical monocytes to inflammation sites that may result in virus acquisition and, in turn, facilitate virus dissemination and viral persistence. Our findings thus provide new insight into the importance of monocyte infection in viral spread and suggest targeting of monocytes for therapeutic intervention.

Palmitoylation and p8-Mediated Human T-Cell Leukemia Virus Type 1 Transmission

ABSTRACT The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.

Role of myosin Va on HTLV-1 p8I protein's traffic to cell surface

HTLV-1 ORF-I encodes the 99 amino acid p12 protein which can be proteolytically cleaved at the amino terminus to generate the p8 protein. The first proteolytic cleavage removes the ER retention/retrieval signal at the amino terminus of p12, while the second cleavage generates the p8 protein. The p12 protein localizes to cellular endomembranes, within the ER and Golgi apparatus, while p8 traffics to lipid rafts at the cell surface and is recruited to the immunological synapse upon T-cell receptor (TCR) ligation. In secretory vesicle transport, vesicles produced as post-Golgi are moved along the cytoskeleton by motor proteins. The unconventional myosin motor, myosin V, moves several cargoes including secretory vesicles, synaptic vesicles, and the endoplasmic reticulum. To understand if p8 traffics from Golgi apparatus to cell surface on a myosin Va dependent manner, Jurkat T cells were transfected with pMEp12 plasmids which express variants (p12WT, p12Δ29 and p12G29S) of the fusion protein of HTLV-1 p12I tagged with the influenza hemagglutinin (HA1) tag and with the Myo Va full-tail neuronal-eGFP conjugated (MyoVa FTNeu-eGFP) plasmid which expresses a negative dominant of myosin Va and competes for intracellular ligands with cellular putative myosin. Both p12/p8 and myosin Va proteins intracellular localization were analyzed by indirect immunofluorescence assay using antibodies against the HA-tag and the Myo Va protein. Confocal microscopy and image collection was performed by using a p12I/p8I proteins Zeiss LSM 780 microscope (Carl Zeiss Optical, Chester, Va.) with Adobe Photoshop CC software. It was reported p12I expression in Jurkat T as previous described was shown in perinuclear region which might be RE and Golgi apparatus and that p12I/p8I and MyoVa proteins colocalizes, however only when MyoVa FTNeu-eGFP was simultaneously expressed with pMEp12 plasmids, p12I/p8I localization showed to be altered from dots dispersed all over cytoplasm and cell surface to form cytoplasmic aggregates independently on variant of p12I expressed, suggesting that myosin Va plays an important role on traffics of p8I from Golgi to cell surface.

HTLV-1 burden dependent on hijacking monocytes chemotaxis

The HTLV-DNA burden in PBMCs is a risk factor for HAM/TSP and ATL development. We investigated the contribution of monocyte subsets (classical, intermediate and non-classical) to the total viral burden in 23 HTLV-1 infected individuals by assessing their frequency, their chemotactic and phagocytic functions, as well as their infectivity status. Classical monocytes differed between infected and uninfected individuals, their frequency was lower and their expression level of the chemokine receptors CCR5, CXCR3 and CX3CR1 was higher. While the percentage and surface chemokine receptor expression did not differ between HTLV-1 infected and uninfected individuals, intermediate monocytes from HTLV-1 infected individuals had increased migratory capacity to CCL5, the ligand for CCR5. Non-classical monocytes from HTLV-1 infected individuals increased in frequency and expressed high levels of CCR1, CXCR3 and CX3CR1. All three purified monocyte subsets were infected by HTLV-1. The level of viral DNA in monocyte subsets correlated directly with their migration capacity to CCL2, CCL5 and CX3CL1 for the classical subset, with lesser phagocytosis for the intermediate monocytes, and with the level of viral DNA in CD8+ and CD4+T-cells for the non-classical subset. These data suggest a model whereby HTLV-1 infection augments classical monocytes migration to tissues resulting in their infection, and non-classical monocytes frequency, resulting in increased transmission of virus to CD4+ and CD8+T-cells. Our data in humans together with prior animal experiments supports the notion that infection of monocytes in vitro is crucial for viral infectivity and persistence in humans, rendering infected monocytes desirable therapeutic targets.

The contribution of monocytes/macrophages to HTLV-1 infection and persistence

Peripheral blood monocytes can be classified into three main subsets: CD14++CD16– (classical), CD14+CD16++ (non-classical), and CD14++CD16+ (intermediate) which exert important roles in innate and adaptive immunity. Here we investigated whether the different monocyte subsets are potential HTLV-1reserviors that contribute to viral infection and persistence. PBMCs from 17 HTLV-1 infected patients (IP) and 11 normal donors (ND) were phenotypically analyzed by flow cytometry. Classical monocyte frequency was lower in HTLV-1-IPs compared to NDs (p=0.048). Moreover, we found a positive correlation between PVL and intermediate monocyte frequency (r=0.6735, p=0.0042). Focusing on the presence of HTLV-1 provirus DNA in the different monocyte subsets, cell populations were isolated from PBMCs of 16 HTLV-1-IP. When we analyzed by nested PCR genomic DNA isolated from sorted CD4+, CD8+ CD14++CD16–, CD14+CD16++, and CD14++CD16+, we found that HTLV-1 patients with high PVL, all monocyte subsets as well as CD4+ and CD8+ cells were positive for HTLV-1. In contrast, the intermediate monocytes were negative or very weakly positive for HTLV-1 in patients with low PVL. To test whether natural STLV-1 infection recapitulates what we find in HTLV-1-IPs, we analyzed the monocyte subsets distribution in 8 STLV-1 infected Rhesus macaques and 16 naive animals. Consistent with human infection, the frequency of intermediate monocytes was higher in infected macaques compared to naive animals (p=0.0001) with a positive correlation between PVL and intermediate monocytes frequency (r=0.6530, p=0.03). In conclusion, our results suggest that monocytes play an important role in viral dissemination and persistence and are potential viral reservoirs.

Both orf-I isoforms, p12 and p8, are required for efficient HTLV-1 infection and persistence

HTLV-1 orf-I is important for immune evasion, viral replication and persistence. Here, we analyzed proviral sequences isolated from ex vivo PBMC samples of 163 HTLV-1-infected patients (85 carriers, 78 HAM/TSP). Of the 5000 sequences analyzed, orf-I expression was consistently maintained. While the highest percentage of isolates express both p12 and p8 (69%), the mutants that express mainly p8 (7%) or mainly p12 (24%) were highly correlated with decreased proviral loads. To determine the individual role of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing either p12(G29S) or p8(N26) and established producer cell lines used to infect macaques. Three of four macaques infected with WT-HTLV-1 tested positive by PCR and were seropositive. Only one of the macaques infected with N26-HTLV-1 tested positive by PCR and two macaques were seropositive. None of the 8 animals infected with G29S-HTLV-1 tested PCR positive or seroconverted. These results suggest that expression of both p12 and p8 are critical for viral persistence. Since orf-I plays a role in T-cell activation and recognition, we compared the CTL responses elicited by CD4+ T-cell infected with the different HTLV-1 clones. Although the supernatant p19 levels and proviral loads for all four infected lines were similar, a significant difference in Tax-specific CTL killing was observed. Cells infected with orf-I-knockout virus, p12-G29S or p8-N26 were killed whereas cells infected with WT virus were not killed by Tax-specific CTLs. These results indicate that efficient viral persistence and spread requires expression of both p12 and p8.

Role of dimerization and palmitoylation on the function of HTLV-1 p12 and p8

HTLV-1 p12 is an endoplasmic reticulum resident protein that is cleaved to generate p8, which traffics to the cell surface. p12 promotes T-cell activation by increasing NFAT activity while p8 induces T-cell anergy and enhances virus transmission. We have previously demonstrated that p12 and p8 coimmunoprecipitate. Though the dimerization domain of p12 and p8 is unknown, these proteins contain a single cysteine residue which may form intermolecular disulfide bonds. We have also determined that p8 localizes to membrane lipid rafts. Of importance, palmitoylation increases the hydrophobicity of proteins to target them to lipid rafts. As palmitoylation occurs on cysteine residues, we hypothesize that this post-translational modification regulates p12 and p8 localization, dimerization, and function. In this study, we have demonstrated that wildtype p12 and p8 formed hetero- and homodimers while a mutation at the cysteine residue (C39A) inhibited dimer formation and that monomeric wildtype p12 and p8, but not the C39A mutant, are pamitoylated. Immunofluorescence analysis showed that wildtype p8 localized at the cell surface while C39A p8 did not. This result suggests that p8 homodimerization or palmitoylation regulates p8 localization. We also analyzed ex vivo DNA samples from HTLV-1-infected individuals and found DNA polymorphisms at position 39. These naturally occurring polymorphisms affected dimerization and localization of p8. Currently, we are investigating whether mutation at C39 affects NFAT activation, virus transmission, or proviral load. Determining the mechanism by which p12 and p8 localization, dimerization, and functions are regulated will add to our understanding of HTLV-1 pathogenesis.

ORFI genetic polymorphisms in geographically distinct HTLV-1 infections

The region known as pX in the 3’ end of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. The expression of ORF-I produces the protein p12 which can be cleavage resulting in the p8 protein. These proteins interfere with the ability of a biologically active of HTLV-1, influencing at the virus infectivity and persistence. Here, we evaluated whether natural mutations in HTLV-1 ORF-I can influence the proviral load and clinical manifestation of HAM/TSP. For that, we analyzed 1530 HTLV-1 ORF-I sequences from different clones of 156 patients with negative or positive diagnosis for HAM/TSP and demonstrated that some mutations may be associated with the outcome of HAM/TSP (C39R, L40F, P45L, S69G and R88K) or with proviral load (P34L and F61L). We further examined the presence of mutations in motifs of HBZ and observed that P45L mutation is located within the HBZ nuclear localization signal and was found more frequently between patients with HAM/TSP and high proviral load. This results suggest that some HTLV-1 ORF-I mutations may be associated with the development of HAM/TSP and the proviral load levels.