ReviewExpression and functional properties of proteins encoded in the x-II ORF of HTLV-I
Introduction
Human T-lymphotropic virus types I and II (HTLV-I and HTLV-II) are members of the HTL/BLV group of retroviruses, together with bovine leukemia virus and simian T-cell leukemia virus type I. These viruses are characterized by a complex genome whose expression is regulated by viral-encoded proteins termed Rex and Tax. Infection with HTLV-I may result in two distinct pathologies, adult T-cell leukemia (ATL), an aggressive malignancy of mature CD4+ T-cells, and tropical spastic paraparesis/HTLV-associated myelopathy (TSP/HAM), a demyelinating neuropathy. In addition, accumulating evidence supports an association between HTLV-I infection and a number of inflammatory diseases of probable autoimmune origin. To date, HTLV-I is the only retrovirus that has been conclusively recognized to play a causative role in human neoplasia; although originally isolated from a patient with hairy cell leukemia, HTLV-II does not appear to be oncogenic in vivo (for a general review of the HTLV/BLV viruses, see Cann and Chen, 1996).
The HTLV/BLV genome is distinguished by the presence of the unique X region containing partially overlapping open reading frames (ORFs). In the case of HTLV-I, these ORFs are termed x-I, x-II, x-III, x-IV and x-V. The major portions of the Rex and Tax proteins are coded by the x-III and x-IV ORFs, respectively. Rex controls the expression of the unspliced and the singly spliced viral mRNAs encoding the structural proteins; this effect is mediated by binding of Rex to viral transcripts via a cis-acting element (RXRE) located in the LTR and by the ability of Rex to inhibit splicing and interact with proteins involved in controlling macromolecular trafficking between the nucleus and the cytoplasm (reviewed by Rabson and Graves, 1997). Tax activates expression of the LTR promoter, and thus is essential for the expression of all viral mRNAs. Tax also activates transcription of a number of cellular genes via interaction with the NF-κB, cAMP-responsive element binding factor, and serum-responsive factor families of transcription factors, and is the major determinant of HTLV-I oncogenicity (reviewed by Yoshida, 1996).
A number of features of the HTLV-I life cycle and pathogenesis are still incompletely understood, including the factors determining whether infection will be either clinically inconsequential or lead to development of ATL or TSP/HAM, and the mechanism underlying the long latency period (up to several decades) between primary infection and disease. In addition, the pathogenetic mechanism leading to neurological damage in TSP/HAM remains to be elucidated, although a CTL-mediated autoimmune mechanism and a correlation with particular HLA haplotypes have been postulated (Jacobson et al., 1990, Jeffery et al., 1999). Given that the virus is able to infect a very wide spectrum of cell types in vitro, it is also unclear why HTLV-I infection is restricted mainly to CD4+ lymphocytes in vivo. Furthermore, although Tax is known to be essential for HTLV-I-mediated transformation and is sufficient to immortalize primary human T lymphocytes (reviewed by Franchini, 1995), it is clear that Tax-transduced T cells do not exhibit a fully transformed phenotype, e.g., they are dependent on exogenous IL-2 for their growth (Akagi et al., 1995), and do not show constitutive activation of the JAK/STAT signal transduction pathway (Migone et al., 1995, Xu et al., 1995), suggesting that additional components, possibly viral genes, might be required for the emergence of a transformed phenotype.
To investigate if any additional viral-encoded products might represent molecular determinants of these processes, we and others have explored the coding potential of the X region in further detail. These studies identified a number of novel alternatively spliced viral mRNAs coding for ORFs x-I, x-II, and x-III detected in HTLV-I-infected cells (Berneman et al., 1992, Ciminale et al., 1992, Koralnik et al., 1992). The coding potential of these messages has been verified in transient transfection systems, and preliminary information regarding the functional properties of their protein products has been obtained (Franchini et al., 1993, Mulloy et al., 1996, D'Agostino et al., 1997, Albrecht et al., 1998, Collins et al., 1998, Collins et al., 1999, Ciminale et al., 1999, Bartoe et al., 2000). This review will focus on our studies on two protein isoforms encoded in the x-II ORF of HTLV-I termed Tof/p30II and p13II.
Section snippets
Expression of ORF x-II proteins
The x-II ORF may be translated from two alternatively spliced mRNAs, giving rise to two protein isoforms differing in their amino terminal portion (Fig. 1). A double splicing event joining exons 1 and 2 to a 3′ splice site that defines exon B gives rise to an mRNA coding for a 241-amino acid protein termed Tof/p30II; translation of Tof is started in exon 2 at the same AUG codon used by Tax. This mRNA has been detected in HeLa cells transfected with the HTLV-I molecular clone CS-HTLV-I and in
Functional properties of Tof/p30II
Initial studies of Tof demonstrated that the protein accumulates primarily in the nucleolus and nucleus with a pattern that is reminiscent of that of the Rex regulatory protein. Tof exhibits an apparent size of 30 kDa in SDS-PAGE gels (Ciminale et al., 1992) and is phosphorylated (D'Agostino, unpublished observations). Comparison of Tof's amino acid sequence with sequence data bases revealed no prominent homologies with the exception of serine clusters resembling the activation domains of
Functional properties of p13II
Initial studies carried out using p13II expression plasmids showed that the protein exhibits an apparent size of 13 kDa (Koralnik et al., 1992) and accumulates primarily in discrete cytoplasmic structures (D'Agostino et al., 1997) (Fig. 2). Further studies by immunofluorescence/dual staining with different marker proteins identified the cytoplasmic structures as mitochondria (Ciminale et al., 1999). Using in vitro mutagenesis, we defined the minimal sequence required for mitochondrial targeting
Conclusions and perspectives
Our understanding of the function and biological significance of Tof/p30II and p13II remains rudimentary. Future studies of Tof/p30II will be aimed at confirming whether the protein influences RNA expression, which will require identification of its RNA target. Additional experiments will be necessary to verify whether the alterations in mitochondrial morphology/architecture observed in p13II-expressing cells represent a prelude to apoptosis or translate into functionally relevant differences
Acknowledgements
We wish to thank Daniela Saggioro for discussions and Pierantonio Gallo for artwork. LZ is a recipient of a fellowship from ANLAIDS. The research described in this report was supported by grants from the Istituto Superiore di Sanita’ (ISS AIDS research program # 9204-28), the Associazione Italiana per la Ricerca sul Cancro (AIRC), and the Fondazione Italiana per la Ricerca sul Cancro (FIRC). The laboratory participated in the concerted action ‘HTLV European Research Network’ (HERN) of the
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