In this project we have two main approaches: Approach 1: To integrate information obtained by functional biochemical studies to infectivity and persistence of HTLV-1 in dendritic cells and T-cells in vitro. Approach 2: To verify the role of orf I and orf II in viral persistence in animal models. The viral genome encodes mRNAs for several non-structural proteins that affect cellular pathways and modulate viral replication. One such protein, p12, encoded by orf I, localizes to the ER and Golgi and cellular membranes. The proteolytic cleavage of p12 dictates its cellular localization and functions. The removal of a non-canonical endoplasmic reticulum (ER) retention/retrieval signal within the amino terminus of p12 is necessary for trafficking to the Golgi apparatus and the generation of a completely cleaved 8 kDa protein. The 8 kDa protein traffics to the cell surface, is recruited to the immunological synapse following T-cell receptor (TCR) ligation, down-regulates TCR proximal signaling and increases viral transmission. The full length form of p12 resides in the ER and interacts with the beta and gamma-c chains of the interleukin-2 receptor (IL-2R), the heavy chain of the major histocompatibility complex (MHC) class I, as well as calreticulin and calnexin. Genetic analysis of ORF-I from ex vivo samples of HTLV-1-infected patients reveals frequent amino acid substitutions within orf-I that inhibit proteolytic cleavage, suggesting that ER associated functions of p12I may be selected in vivo. Because HTLV-I orf-I is important for viral transmission and persistence, we sought to determine the genetic variation of p12 protein in HTLV-1 infected individuals and investigate a possible association between p12 mutations, its cleavage status, proviral load, and disease outcome. In this study, we performed reverse genetics of 160 patients to identify mutations in orf-I. We found that individuals that harbor clones expressing both p12 and p8 have higher virus loads compared to individuals that harbor clones expressing predominantly p12 or predominantly p8. Further, we constructed infectious molecular clones expressing distinct orf-I isoforms (p12/p8;p12;p8). Clonal cell lines were established, characterized and used in a rhesus macaque model. Results from these studies clearly demonstrate that viral persistence requires expression of both p12 and p8 isoforms. Importantly, our results suggest that p12/p8 expression impacts CTL escape.We also analyzed the role of in p12 and p8 function. We found that HTLV-1 p12 and p8 form disulfide-linked homo-and heterodimers and that the monomeric forms of these proteins are palmitoylated. Mutation of cysteine 39 within these proteins disrupted dimerization and palmitoylation of both p12 and p8 without affecting protein localization. These studies suggest palmitoylation of p8 dirupts dimer formation and targets the monomeric form to modulate cell signaling pathways to increase cell-to-cell adhesion and viral infectivity. Determining the mechanism by which p12 and p8 dimerization and functions are regulated would advance our understanding of HTLV-1 pathogenesis and could identify potential novel therapeutic targets for the treatment of HTLV-1-infected individuals.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005645-23
Application #
8552582
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2012
Total Cost
$1,780,586
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Rende, Francesca; Cavallari, Ilaria; Andresen, Vibeke et al. (2015) Identification of novel monocistronic HTLV-1 mRNAs encoding functional Rex isoforms. Retrovirology 12:58
Cavallari, Ilaria; Rende, Francesca; Bona, Marion K et al. (2015) Expression of Alternatively Spliced Human T-Cell Leukemia Virus Type 1 mRNAs Is Influenced by Mitosis and by a Novel cis-Acting Regulatory Sequence. J Virol 90:1486-98
Edwards, Dustin; Fukumoto, Risaku; de Castro-Amarante, Maria Fernanda et al. (2014) Palmitoylation and p8-mediated human T-cell leukemia virus type 1 transmission. J Virol 88:2319-22
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Andresen, Vibeke; Pise-Masison, Cynthia A; Sinha-Datta, Uma et al. (2011) Suppression of HTLV-1 replication by Tax-mediated rerouting of the p13 viral protein to nuclear speckles. Blood 118:1549-59
Edwards, Dustin; Fenizia, Claudio; Gold, Heather et al. (2011) Orf-I and orf-II-encoded proteins in HTLV-1 infection and persistence. Viruses 3:861-85
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Van Prooyen, Nancy; Gold, Heather; Andresen, Vibeke et al. (2010) Human T-cell leukemia virus type 1 p8 protein increases cellular conduits and virus transmission. Proc Natl Acad Sci U S A 107:20738-43

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