---
id: "edwards-2011-orf-encoded"
title: "Orf-I and Orf-II-Encoded Proteins in HTLV-1 Infection and Persistence"
authors:
  - "Dustin Edwards"
  - "Claudio Fenizia"
  - "Heather Gold"
  - "Maria Fernanda de Castro-Amarante"
  - "Cody Buchmann"
  - "Cynthia A. Pise-Masison"
  - "Genoveffa Franchini"
venue: "Viruses"
year: 2011
date: "2011-06-17"
doi: "10.3390/v3060861"
url: "/research/publications/10-3390-v3060861/"
pdf: "/research/publications/10-3390-v3060861/dustin-edwards-10-3390-v3060861.pdf"
pmc: "https://pmc.ncbi.nlm.nih.gov/articles/PMC3185781/"
openAccess: true
license: "cc-by"
citedBy: 52
citedBySource: "OpenAlex, read 2026-09-12"
---
# Orf-I and Orf-II-Encoded Proteins in HTLV-1 Infection and Persistence

Review of the HTLV-1 orf-I and orf-II proteins p12, p8, p13 and p30.

## Abstract

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.

## Full text

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Viruses 2011, 3, 861-885; doi:10.3390/v3060861
viruses
ISSN 1999-4915
www.mdpi.com/journal/viruses
Review
Orf-I and Orf-II-Encoded Proteins in HTLV-1 Infection and
Persistence
Dustin Edwards, Claudio Fenizia, Heather Gold, Maria Fernanda de Castro-Amarante,
Cody Buchmann, Cynthia A. Pise-Masison and Genoveffa Franchini *
Animal Models and Retroviral Vaccines Section, Center for Cancer Research, National Cancer
Institute, National Institutes of Health, Bethesda, Maryland, MD 20892, USA;
E-Mails: edwardsd2@mail.nih.gov (D.E.); feniziac@mail.nih.gov (C.F.); goldhl@mail.nih.gov (H.G.);
amarantem@mail.nih.gov (M.F.d.C.-A.); buchmannc@mail.nih.gov (C.B.);
masisonc@mail.nih.gov (C.A.P.-M.)
* Author to whom correspondence should be addressed; E-Mail: franchinig@mail.nih.gov;
Tel.: +1-301-496-2386; Fax: +1-301-402-0055.
Received: 13 April 2011; in revised form: 25 May 2011 / Accepted: 26 May 2011 /
Published: 17 June 2011
Abstract: 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.
Keywords: human T-cell leukemia/lymphoma virus type-1; HTLV-1; ORF-I; ORF-II; p8;
p12; p13; p30
OPEN ACCESS

Viruses 2011, 3 862
1. Introduction
Human T-cell leukemia/lymphoma virus type-1 (HTLV-1) is an oncogenic retrovirus first
discovered in 1980 in T-cells of a patient with cutaneous T-cell lymphoma [1,2]. HTLV-1 is the
etiological agent of two major diseases: adult T-cell leukemia (ATL), a disease characterized by
malignant proliferation of CD4+ T-lymphocytes, and tropical spastic paraparesis/HTLV-1-associated
myelopathy (TSP/HAM), a neurodegenerative condition [3,4]. HTLV-1 is also associated with other
clinical disorders including HTLV-1-associated arthropathy, HTLV-1-associated uveitis, infective
dermatitis, and polymyositis [5,6]. HTLV-1 primarily infects CD4+ T-cells and has been detected in
ex vivo CD8+ T-cells, dendritic cells (DC), and B-cells from infected individuals. While cell-free
virions have been shown to efficiently infect DCs in vitro, HTLV-1 is believed to be transmitted to
T-cells and DCs mostly by cell-to-cell contact through a virological synapse, biofilm-like extracellular
viral assemblies, or cellular conduits [7–10]. An estimated 10–20 million people worldwide are
infected with HTLV-1 [11]. While the majority of HTLV-1-infected individuals remain asymptomatic,
a low percentage of patients develop either ATL (3–5%) or TSP/HAM (0.3–2%) after a long period of
clinical latency [12–19].
As shown in Figure 1, the HTLV-1 genome contains the typical retroviral structural and enzymatic
genes gag, pro, pol, and env [13]. In addition, a region located between env and the 3' long terminal
repeat (LTR), contains four partially overlapping open reading frames (ORF) [13]. This unique region
encodes several regulatory proteins through the use of alternative splicing and internal initiation
codons [20–22]. Orf-I produces the p12 protein which can be proteolytically cleaved at the amino
terminus to generate the p8 protein, while differential splicing of mRNA from orf-II results in
production of the p13 and p30 proteins [20–23]. Orf-III and orf-IV encode for the Rex and Tax
proteins, respectively, and an antisense mRNA transcribed from the 3' LTR that generates the HTLV-1
basic leucine zipper (HBZ) protein [24–26].
Tax and Rex are required for viral replication. Tax is a potent transcriptional transactivator of viral
gene expression. Tax also regulates the expression of several cellular genes, including those involved
in cell proliferation, cell cycle progression, apoptosis, and DNA damage responses. Rex is a
post-transcriptional regulator that facilitates nuclear export of unspliced and singly spliced viral
mRNA. In addition, Rex inhibits splicing and transport of doubly spliced mRNA. HBZ is a negative
regulator of Tax-mediated transactivation and thus suppresses viral expression. For further detailed
information about Rex, Tax, and HBZ, the reader is referred to recent reviews [27–30]. In this review,
we will focus on the current knowledge of the functions of the proteins encoded by orf-I and orf-II: p8,
p12, p13, and p30.
In contrast to Tax and Rex, orf-I and orf-II are dispensable for viral replication in vitro yet are
important for viral persistence in vivo [31]. Early work demonstrated that in the rabbit model, orf-I was
required for viral infectivity while orf-II was required to maintain high viral load [32,33]. Further work
in the rabbit model showed reversion of HTLV-1 clones lacking p30 to the wildtype p30-expressing
virus, suggesting the importance of p30 to HTLV-1 viral persistence [34]. However, in these early
studies the HTLV-1 clones that were used contained a frameshift that affected hbz, making it unclear
as to whether these effects were due to the loss of hbz or orf-I and orf-II-encoded proteins. In a more
recent study, the ablation of p12/p8, p30, or HBZ impaired the establishment of persistent infection in

Viruses 2011, 3 863
the macaque model [35]. Nevertheless, ablation of these proteins did not affect viral replication in the
rabbit model [35]. The orf-I and orf-II-encoded proteins are able to modulate a diverse range of viral
and cellular mechanisms including transcriptional regulation, mitochondrial function, cell cycle
progression, host cell activation and proliferation, apoptosis, virus infectivity and transmission, and
host immune responses. Though these proteins are not essential for virus replication in vitro, p8, 12,
p13, and p30 have an important role in the establishment and maintenance of HTLV-1 infection in vivo.
Figure 1. A scheme of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1)
genome. Spliced mRNAs and encoded proteins for orf-I and orf-II are shown. Orf-I
encodes for the p12 protein which can be proteolytically cleaved at the amino terminus to
generate the p8 protein. The p30 protein is translated from doubly spliced mRNA
transcribed from orf-II and the 5' end of env. The p13 protein is translated from singly
spliced mRNA transcribed from orf-II and corresponds to the carboxyl terminus of p30.
2. HTLV-1 p12 and p8
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 (Figure 1). Computational analysis of the amino acid
sequence of p12 predict the existence of a noncanonical endoplasmic reticulum (ER) retention/retrieval
signal between amino acids 1–5, two putative leucine zipper (LZ) motifs, two putative transmembrane
domains between amino acids 12–30 and amino acids 48–67, a calcineurin-binding motif between amino
acids 70–86, four putative proline-rich (PXXP) Src homology 3 (SH3)-binding domains, and a
putative adaptin motif [23,36]. These structural features may contribute to protein localization,
homodimerization, and protein-protein interactions. The p12 protein exhibits amino acid similarity
with a portion of the bovine papillomavirus (BPV)-transforming E5 protein, except that E5 does not
carry putative SH3 binding motifs [37,38]. The p12 protein undergoes complex post-translational
modifications through proteolytic cleavage. The first cleavage occurs between amino acid positions 9

Viruses 2011, 3 864
and 10 and is followed by a second cleavage between amino acids 29 and 30 [23]. The first proteolytic
cleavage removes the ER retention/retrieval signal at the amino terminus of p12, while the second
cleavage generates the p8 protein [23]. The p12 protein localizes to cellular endomembranes,
particularly 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 [23,39–41].
The singly spliced mRNA encoding p12/p8 has been detected in vitro and in ex vivo HTLV-1-infected
T-cells and macrophages [42]. The p12 recombinant protein is recognized in serum from humans
infected with HTLV-1 and rabbits experimentally infected with HTLV-1 [43]. In addition, a cytotoxic
T-lymphocyte (CTL) response to orf-I products can be detected in HTLV-1-infected individuals [44].
Two natural variants of the p12 protein have been identified; one variant carries a lysine residue at
position 88 and is commonly found in HTLV-1 strains from TSP/HAM patients while the second
variant carries an arginine residue at position 88 and is found in HTLV-1 strains from all ATL patients
and healthy carriers studied. The R88 variant protein has a much greater stability compared to the K88
variant, which is ubiquitinated and rapidly degraded by the proteasome [45].
2.1. T-Cell Signaling
2.1.1. Calcium Release
The p12 protein resides in the endoplasmic reticulum, which has a role in protein and lipid
synthesis, carbohydrate metabolism, and calcium concentration regulation. Within the ER, p12 is able
to mediate an increase in cytosolic calcium in T-cells by increasing calcium release from the ER
through inositol trisphosphate receptors and from capacitative calcium entry through Ca2+ channels at
the plasma membrane in response to the lower ER calcium content (Figure 2(1)) [46,47]. By depleting
ER calcium stores and increasing cytosolic calcium, p12 is able to modulate a range of processes
including T-cell proliferation, viral replication, and viral spread. Early studies on orf-I showed that p12
is able to activate nuclear factor of activated T-cells (NFAT), which is dependent on calcium-binding
proteins for its dephosphorylation and nuclear import, to increase T-cell proliferation (Figure 2(2))
[46–48]. Furthermore, p12 can impact other calcium-regulated proteins, including the transcriptional
coactivator p300, which can modulate transcription of viral genes from the HTLV-1 LTR [49,50].
Moreover, p12 can promote cell-to-cell viral spread by inducing lymphocyte function-associated
antigen 1 (LFA-1) clustering on T-cells through a calcium-dependent mechanism (Figure 2(5)) [51].
2.1.2. NFAT Activation and Signaling
Prior to the discovery of p12 cleavage, expression of orf-I-encoded proteins (ORF-I) was shown to
enhance T-cell proliferation [46,51,52]. Early studies found that ORF-I was able to mediate activation
of NFAT, a transcription factor that regulates activation, proliferation, and differentiation of T-cells
[46–48]. In uninfected cells, NFAT can be activated through a complex TCR signaling cascade.
Following TCR engagement at the cell surface, the protein tyrosine kinases Lck and Fyn phosphorylate
TCRδ and the CD3 subunits. These phosphorylated domains become the docking sites for ZAP70.
Activated ZAP70 phosphorylates linker for activation of T-cells (LAT), which then binds and activates
phospholipase C-γ-1 (PLCγ1), leading to the production of inositol-1,4,5-trisphosphate and release of

Viruses 2011, 3 865
Ca2+ from ER calcium stores. The increase in cytosolic calcium activates calmodulin and calcineurin,
which dephosphorylate NFAT, allowing for NFAT nuclear import. As discussed in Section 2.1.1, by
modulating the regulation of cytosolic calcium levels, p12 is able to mediate NFAT activation and
does so independent of the proximal TCR signaling molecules, LAT and PLCγ1 (Figure 2(2)) [47].
Interestingly, p12 is able to bind calcineurin and the calcineurin-binding motif of p12 is homologous to
the calcineurin-binding motif of NFAT (Figure 2(2)) [48]. Thus, p12 could both enhance and inhibit
NFAT activation by competing with NFAT for calcineurin binding. Further studies found that p8,
which localizes at the cell surface, is also able to downregulate NFAT activity, but in a LAT-dependent
manner (Figure 2(3)) [52].
2.1.3. Proximal T-Cell Signaling and T-Cell Anergy
Recent studies indicate that p8 decreases T-cell activation by inhibiting proximal T-cell receptor
signaling [52]. Upon ligation of the TCR to the major histocompatibility complex class II (MHC-II) of
an antigen presenting cell, p8 localizes to the immunological synapse where it decreases
phosphorylation of LAT, PLCγ1, and Vav by a LAT-dependent mechanism (Figure 2(3)) [23,52].
By dampening TCR signaling, p8 downregulates NFAT activation, a crucial pathway in T-cell
activation [47,52]. Furthermore, the induction of T-cell anergy, a state in which T-cells become
unresponsive to TCR stimulation, results in decreased Tax activity and HTLV-1 replication [52].
Lastly, since it has been recently shown that p8 transfers to neighboring cells, it is possible that
p8-induced T-cell anergy allows a safe transfer of the virus to target cells [9]. These results may
underlie the finding that HTLV-1-infected individuals experience some immune deficiency and are
susceptible to opportunistic infections [53,54].
2.1.4. IL-2 Receptor Activation and STAT5 Signaling
HTLV-1-infected T-cells proliferate in the absence of IL-2 and this IL-2 independence correlates
with constitutive activation of the Janus-associated kinase and signal transducer and activator of
transcription (JAK-STAT) pathway, a transcription factor cascade that affects cell proliferation,
differentiation, and apoptosis [55]. Early work showed that ORF-I did not have a role in IL-2
independence since it did not affect expression of the interleukin-2 receptor (IL-2R) or IL-2
responsiveness [56]. Also, expression of ORF-I did not affect phosphorylation of JAK-STAT proteins
[56]. However, more recent studies have demonstrated that ORF-I binds the β and γc chains of the
immature IL-2R [57]. This interaction stabilizes the IL-2R β and γc chains in a pre-Golgi compartment
and prevents their trafficking to the plasma membrane, leading to a decrease in IL-2R at the cell
surface [57]. Specifically, ORF-I binds the 20 amino acid region proximal to amino acid 350 of the
IL-2R β chain that is critical for JAK1 and JAK3 recruitment, which occurs after IL-2 signaling [58].
The interaction of ORF-I and IL-2R leads to an increase in STAT5 phosphorylation and DNA binding
activity in the absence of IL-2 [58]. This effect is dependent on the presence of the β and γc chains and
JAK3 [58]. By binding the IL-2R, ORF-I decreases the requirement of IL-2 for proliferation in T-cells
in the presence of suboptimal antigen stimulation [58].

Viruses 2011, 3 866
2.2. MHC-I Degradation
HTLV-1 modulates T-cell activation and, in addition, has evolved mechanisms to avoid immune
recognition of infected cells. On the cell surface, MHC-I complexes present peptides to TCRs of
cytotoxic T-lymphocyte. In the case of virus-infected cells, this interaction leads to recognition of viral
peptides and destruction of infected cells. Prevention of MHC-I expression is useful to a number of
viruses to maintain the balance between the host and pathogen. Adenovirus E19 protein can retain
MHC-I in the ER by interacting with the 1 and 2 regions of class I heavy chains through a dilysine
motif [59]. HCMV type I membrane glycoproteins US2 and US11 target MHC-I heavy chains for
degradation by the proteasome [60]. Additionally, HIV Nef and Vpu proteins accelerate endocytosis of
MHC-I complexes and bind to and destabilize newly synthesized MHC-I, respectively [61–63]. In the
ER, p12 binds to newly synthesized MHC-I heavy chains and prevents them from associating with the
β2-microglobulin, a component of the mature MHC-I complex (Figure 2(4)) [40]. Since improperly
assembled proteins are removed from the ER for degradation, the p12-mediated inhibition of MHC-I
heavy chain association with the β2-microglobulin leads to its degradation by the proteasome and
results in decreased MHC-I cell surface expression. By decreasing antigen presentation through
degradation of the MHC-I, p12 may diminish presentation of viral peptides and decrease recognition
by cytotoxic T-lymphocytes.
2.3. Modulation of ICAM
Natural killer (NK) cells recognize and destroy cells that express low levels of MHC-I at the cell
surface. ORF-I decreases MHC-I expression to inhibit presentation of viral proteins to cytotoxic
T-lymphocytes, which could make HTLV-1-infected cells susceptible to NK cell cytotoxicity [40,64].
In contrast, HTLV-1-infected T-cells are resistant to NK cell-mediated killing [64]. This resistance can
be moderately ameliorated by pretreatment of NK cells with IL-2 [64]. Early data demonstrated that
several ATL cells lines had altered expression of intercellular cell adhesion molecule 1 (ICAM-1), a
glycoprotein that facilitates the interaction between NK cells and T-cells [65]. Furthermore, the
majority of HTLV-1-infected primary CD4+ T-cells do not express ligands for the NK cell activating
receptors, natural cytotoxicity receptors, and NKG2D [64]. Recent work has elucidated these findings
as it is now known that ORF-I decreases expression of ICAM-1 and ICAM-2, but not ICAM-3, in
T-cells. Thus, ORF-I inhibits NK cell adhesion to T-cells and prevents virus-infected cells from being
recognized in the presence of low levels of MHC-I [64].
2.4. V-ATPase
The BPV E5 oncoprotein interacts with the 16 kDa subunit of the H+ vacuolar ATPase (V-ATPase),
resulting in alkalization of the Golgi apparatus [66,67]. The sequence homology between HTLV-1
ORF-I and BPV E5 led to the demonstration that p12 interacts with 16 kDa subunit of the V-ATPase
[68,69]. The transmembrane domains of ORF-I appear to be dispensable for binding to the V-ATPase,
while conservation of the proline-rich domains between amino acids 36 and 48 contributes to the
strength of this interaction [37,69]. The 16 kDa protein is a membrane component of the V-ATPase,
which is also found in clathrin coated vesicles, lysosomes, endosomes, Golgi vesicles, endoplasmic

Viruses 2011, 3 867
reticulum, and synaptic vesicles. This proton pump is responsible for the acidification of these
intracellular vesicles [70]. The atypical function of the proton pump through binding of viral proteins
such as HTLV-I p12 and BPV E5 proteins may interfere in functions like the dissociation of
receptor-ligand complexes and trafficking within the endosomal/lysosomal compartment. In addition,
the acidification is essential for the formation of endosome carrier vesicles, which are intermediates
between early and late endosomes [71,72]. HTLV-1 is known to infect dendritic cells and the
acidification of lysosomes could play an important role in virus entry [7,73,74]. Indeed, the ablation of
ORF-I expression impairs HTLV-1 replication in dendritic cells [35].
Figure 2. Functions of p12 and p8. In the ER, p12 is proteolytically cleaved at the amino
terminus to generate p8, which traffics to the cell surface through the secretory pathway.
(1) In the ER, p12 mediates Ca2+ release, which enables (2) either calcineurin binding of
NFAT and subsequent dephosphorylation, nuclear translocation, and upregulation of the
IL-2 gene, or p12 binding to calcineurin and inhibition of NFAT activation. (3) Upon
trafficking through the secretory pathway, p8 localizes at the immunological synapse
where it interacts with LAT and inhibits proximal TCR signaling. (4) In the ER, p12 binds
the immature heavy chains of the MHC-I and prevents their interactions with the
β2-microglobulin, leading MHC-I degradation by the proteosome. (5) At the cell surface,
p8 increases the clustering of LFA-1 and the formation of intracellular conduits and
facilitates viral transmission to target cells.

Viruses 2011, 3 868
2.5. Modulation of Virus Transmission in vitro and in vivo
HTLV-1 requires orf-I in vivo to establish a persistent viral infection [33,35]. Early studies reported
that orf-I expression was necessary for HTLV-1 infection in the rabbit model [33]. However, these
early studies used HTLV-1 clones that, in addition to deleting orf-I, produced a frameshift affecting
the gene encoding HBZ. Therefore, it is unclear whether these results are due to deletion of hbz, orf-I,
or both. More recently, HTLV-1 molecular clones with nucleotide mutations were used to selectively
disrupt orf-I expression. This study shows orf-I is essential for infectivity in the macaque model but
not in the rabbit model [35]. Orf-I expression in HTLV-1-infected T-cells enhances virus transmission
to target cells [75]. Independent of IL-2, ORF-I increases chemotaxis to facilitate the migration of
infected cells toward target cells [75]. Importantly, HTLV-1 infection requires cell-cell contact for
efficient transmission through a virological synapse, biofilm-like extracellular viral assemblies, or
cellular conduits [8–10,76]. Transfer of virus between cells at the virological synapse requires
polarization of cytoskeletal proteins and adhesion molecules toward the site of cellular contact [8].
Recent evidence suggests that p8, one of the two orf-I products, modulates the clustering of the
adhesion molecule LFA-1 to increase the formation of cell-cell contacts and facilitate virus transfer
(Figure 2(5)) [9,51]. In addition, p8 promotes the formation of thin membranous cellular conduits,
which allows intracellular communication between several cell types [9,77,78]. Through these
conduits, the HTLV-1 proteins p8, Gag, and Env are transferred to target T-cells [9]. Altogether, p8
promotes cellular contacts to favor HTLV-1 transmission.
3. HTLV-1 p30
Initially identified in 1992, the p30 protein is translated from doubly spliced monocistronic mRNA,
containing exons 1, 2, and B, transcribed from HTLV-1 orf-II (Figure 1) [20,21,79]. p30 is a highly
basic protein with a net positive charge that contains three nuclear localization signals (NLS1, NLS2,
and NLS3) located between amino acids 66–73, 91–98, and 200–241 and an arginine-rich nucleolar
localization/retention (NoRS) domain between amino acids 73–78 [80]. p30 also contains a
Rex-binding domain (RexBD) between amino acids 131–164, a p300-binding domain between amino
acids 1–132, and a DNA-binding domain between amino acids 100–179 [81]. The DNA-binding
domain has been shown to repress LTR-mediated transcription [82]. Notably, HTLV-1 p30 has low
genetic variability and is similar to HTLV-2 p28, suggesting a conserved mechanism for negative
modulation of virus replication [79,83,84]. p30 shares distant similarities with some human serine-rich
transcriptional activators such as Oct-1, Oct-2, Pit-1, and POU-M1 [22]. The p30 protein localizes
within the nucleus and nucleolus. p30 shows high mobility within the nucleus, yet it is strongly
retained in the nucleolus. Specifically, p30 is located in a granular component where ribosome
subunits are assembled and de novo mRNA is produced. This localization is consistent with the ability
of p30 to bind the ribosomal subunit L18a and to retain in the nucleus the newly transcribed tax/rex
mRNA (Figure 3(1)) [80]. Since L18a and the eukaryotic initiation factor 3 facilitate re-initiation of
translation in the cytoplasm, it is possible that p30 translocates from the nucleoli to the cytoplasm [80].
Similarly, p30 is specifically delocalized from the nucleoli to the nucleoplasm upon DNA damage to
interfere with DNA repair processes [85]. In addition, p30 nucleolar retention signal mutants have
similar functionality as wildtype p30, which raises questions about the function of nucleolar

Viruses 2011, 3 869
localization. A recent hypothesis suggests that p30 retention within nucleoli may serve as a reservoir
for when the protein is needed in the nucleus [85]. Interestingly, it has recently been shown that hbz
mRNA can regulate the production of p30, probably acting as an antisense RNA to silence its
expression (Figure 3(4)) [86]. Localization of p30 within the nucleus and nucleolus suggest that this
protein may mediate critical cellular processes such as cell cycle progression, DNA repair, and mRNA
export [87]. Though the main cellular target of HTLV-1 is CD4+ T-cells, the virus is able to infect
CD8+ T-cells, B-cells, macrophages, and dendritic cells. Intriguingly, recent studies have shown that in
a macaque model, ablation of p30 within the HTLV-1 provirus severely affects infectivity and leads to
reversion of the virus to the wild type genotype [35]. This observation has been confirmed in vitro by
p30 knockout in HTLV-1-infected human primary and monocyte-derived dendritic cells, in
which infection is not sustained over time. In T-cells, p30 is not required in vitro for efficient viral
replication [35].
3.1. Inhibition of Nuclear Export of Tax/Rex mRNA
In contrast to HTLV-1 Tax and Rex, which enhance viral replication, p30 promotes virus latency by
retaining tax/rex mRNA within the nucleus to prevent its export to the cytoplasm (Figure 3(5)) [88].
By downregulating Tax and Rex production, p30 suppresses viral replication. p30 interacts with the
p30 mRNA-responsive element (p30RE) of tax/rex mRNA and with Rex at the RexBD [89].
Interestingly, p30RE spans the exon junction created after env mRNA is spliced, hence p30 binds
spliced tax/rex mRNA but not the unspliced and singly spliced viral RNA [81]. The interplay between
Rex and p30 is a regulatory switch between viral replication and latency. Rex binds with high affinity
to the Rex-responsive element (RexRE) at the 3' end of viral mRNA and, together with CRM1, shuttles
unspliced gag/pol and singly spliced env transcripts to the cytoplasm (Figure 3(3)). Once bound to
mRNA, Rex is no longer accessible to binding by p30 and is able to shuttle transcripts to the
cytoplasm. However, viral mRNA-bound p30 efficiently interacts with Rex, but tax/rex transcripts are
still retained in the nucleus (Figure 3(5)) [89]. Nuclear retention of viral mRNA is reversed by an
excess of Rex, which displaces p30 from the p30RE. A further spliced version of tax/rex has been
found in HTLV-infected cells, p21rex. During splicing of p21rex, the p30RE is removed from rex
mRNA, which allows the transcript to escape p30-mediated nuclear retention. However, the function
of p21rex is still unknown [88,90,91]. Thus, by retaining tax/rex mRNA, p30 decreases the translation
of these two positive regulator of viral replication and promotes latency to escape host immune
surveillance and to favor propagation through cell division and clonal expansion of infected cells.
3.2 Repression of the CRE Pathway
In addition to its posttranscriptional activity, p30 has been shown to function as either a
transcriptional activator or repressor. The ability of p30 to induce transcriptional activation in vitro is
CBP/p300-dependent [92]. CBP/p300 are known binding partners of CREB and Tax and are required
for strong activation of the viral LTR [92]. Several other cellular and viral proteins bind CBP/p300,
including members of the Jun-family, c-Myb, c-Fos, STAT1/2, NF-κB, p53, and TATA-binding
protein (TBP) [82]. p30 disrupts CREB-Tax-p300 complex formation on the TRE (Tax-responsive
element) of the viral LTR, resulting in repression of HTLV-1 transcription [82,93]. p30 has also been

Viruses 2011, 3 870
reported to differentially regulate transcription from the viral TRE and cellular CREB-responsive
elements (CRE) in vitro and in vivo independent of Tax expression [94,95]. While p30 has been shown
to repress CRE-driven gene expression in a dose-dependent manner, low concentrations of p30
enhance LTR activity [95,96]. Therefore, the expression level of p30 may play an important role in its
function in the cell. Because p30 suppresses Tax production by retaining tax/rex mRNA in the nucleus,
it affects CRE- and TRE-mediated transcriptional activation [88]. Interestingly, histone acetyltransferase
(HAT) activity of p300 modulates p30-dependent transcriptional downregulation, whereas p30-dependent
LTR repression is enhanced by deacetylation and inhibited by acetylation [82,93].
Figure 3. Functions of p30. (1) Alternatively double spliced mRNA is translated to form
the Tax and Rex regulatory proteins. (2) Tax protein localizes to the nucleus to exert its
function on the LTR as a positive regulator of viral transcription. (3) Within the nucleus,
Rex recognizes the Rex-responsive elements (RexRE) of viral mRNA and shuttles these
transcripts to the cytoplasm while inhibiting splicing processes. However, some of the viral
RNA is processed in the spliced env mRNA, the double spliced p30, (4) and the
alternatively spliced tax/rex mRNA. p30 mRNA is subject to negative regulation by hbz
mRNA. Once p30 protein is produced, it translocates to the nucleus and (5) interacts with
p30-responsive elements (p30RE) created by the double splicing and therefore is present
on tax/rex mRNA only. Moreover, p30 interacts with Rex to inhibit Rex-mediated nuclear
export of double spliced viral mRNA, including tax transcripts. By preventing Tax
production, p30 decreases viral transcription.

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3.3. Transcriptional and Posttranscriptional Regulation
Microarray gene expression analyses of human T-cells showed that HTLV-1 p30 affects a number
of cellular genes at the transcriptional level. p30 alters expression of a variety of gene families
including those that have a role in transcription, translation, cell cycle progression, DNA replication
and repair, cell signaling, angiogenesis, cell migration, and apoptosis [97]. Furthermore, p30 is able to
retain some cellular transcripts within the nucleus, similarly to viral tax/rex mRNA. Included among
these transcripts are MDM4, which is a regulator of p53, and HDAC3, which is a histone deacetylases
involved in transcriptional repression [97]. Moreover, p30 has been shown to interact with the cellular
transcription factor PU.1 in human macrophages. PU.1 is involved in a variety of cellular pathways
including signal transduction by Toll-like receptor-4 (TLR-4) [98]. The interaction of p30 and PU.1
leads to inhibition of the DNA binding and transcriptional activity of PU.1. This, together with the
p30-mediated inhibition of GSK-3β, yields decreased expression of TLR-4 at the cell surface, resulting
in decreased secretion of pro-inflammatory cytokines, such as MCP-1, TNF-α and IL-8, and an
increase of the anti-inflammatory cytokine IL-10 [98].
3.4. Cell Cycle and DNA Repair
Expression of p30 results in the accumulation of T-cells in the G2 phase of the cell cycle. p30 is
able to enhance phosphorylation and activation of check point kinase 1 (Chk1). Chk1 is activated by
ATM/ATR kinase following single strand DNA damage and results in a G2 arrest of the cell cycle
[99]. p30 specifically binds to ataxia-telangiectasia mutated (ATM) and regulator of 20S proteasome
activators γ (REGγ) in multiprotein high molecular weight complexes. By binding to ATM, p30
prolongs cell survival following DNA damage by inhibiting ATM autophosphorylation and subsequent
activation of proteins involved in DNA repair, cell cycle check points, and apoptosis [100]. The effect
of p30 interactions with REGγ remains to be determined. REGγ promotes formation of the REG-20S
proteasome complex and has a critical role in a number of cellular processes, such as cell cycle
progression and transcriptional regulation, and can degrade proteins in a ATP- and ubiquitin-independent
manner. REGγ is localized in the nucleus where it interacts with and stabilizes p30, thereby altering
p30 turnover [100]. In addition, p30 is able to bind to and prevent complex formation of cyclin E and
cyclin-dependent kinase 2 (CDK2). Disruption of this complex prevents phosphorylation of
retinoblastoma and subsequent E2F-mediated transcription, two key steps for G1/S transition [101]. By
inhibiting cyclin E-CDK2 complex formation, p30 delays entry of cells into the S phase of the cell
cycle. The inhibition of cell cycle progression is consistent with the observation that dendritic cells
isolated from peripheral blood of ATL patients are unable to stimulate proliferation of CD4+ and CD8+
T-cells [102]. p30 is also able to interact with multiple proteins involved in DNA repair processes [85].
Upon DNA damage, p30 specifically delocalizes from the nucleolus to interact with and affect correct
assembly of MRN complexes (Mre11-Rad50-Nbs1). MRN complexes are a key factor of DNA repair
and contribute to homologous recombination (HR) during the S phase of the cell cycle. In contrast,
during G1 or M phases, DNA damage is preferentially repaired by the nonconservative nonhomologous
end joining (NEHJ) pathway. By interacting with these complexes, p30 activates a shift from
conservative HR to the error-prone NEHJ pathway, thus favoring an accumulation of genomic

Viruses 2011, 3 872
alterations. Overall, these observations suggest that p30 could contribute to the accumulation of
mutations that are characteristic of transformed HTLV-1-infected T-cells.
3.5. Requirement of orf-II in Viral Persistence in Animal Models
Despite the effort of multiple laboratories to define the role of HTLV-1 p30, its exact function and
relevance in vivo still remains elusive. It has been previously shown in vitro that the ablation of p30
expression does not affect viral infectivity of HTLV-1 in human primary cells and that p30 is
dispensable for viral replication and immortalization of primary human T-lymphocytes [31,103]. It
should be noted that the in vitro infectivity of p30-ablated HTLV-1 was not sustained over time in
primary dendritic cell [35]. Other observations, such as the presence of antibodies to p30 during
infection may provide evidence of the importance of p30 in vivo [32,104]. It was first shown that the
ablation of p30 expression in vivo result is a dramatic decrease of HTLV-1 viral load in a rabbit model
[32]. However, the mutation introduced to prematurely stop the translation of p30 affected hbz as well,
whose ablation alone decreases viral replication [105]. By inserting an artificial 24 base pair linker
containing a premature termination codon in the p30 ORF, a significant frameshift occurred in the
antisense hbz ORF [32]. The consequence of this frameshift is unknown, but it most likely affected hbz
expression. Later studies have used different mutations to ablate p30 expression while preserving
hbz [35]. These in vivo studies showed that p30 is not necessary for HTLV-1 infectivity in a rabbit
model as viral replication was not affected by p30 ablation [35]. In contrast, the mutation of p30
severely affects virus infectivity in macaques, although a sufficient level of viral replication occurred
to allow the reversion to wild type p30 over time [35].
4. HTLV-1 p13
Differential splicing of mRNA from HTLV-1 orf-II results in production of the p13 protein
[20–22,106]. p13 is translated from singly spliced monocistronic mRNA to form a highly basic 87
amino acid protein that corresponds to the carboxyl terminus of p30 (Figure 1) [21]. The p13 protein
has been predicted to contain a short hydrophobic leader sequence between amino acids 1–5, an amino
terminus mitochondrial targeting signal (MTS) in a positively charged amphipathic alpha helix
between amino acids 22–31, a transmembrane domain between amino acids 30–40, a flexible hinge
region between amino acids 42–48, and a carboxyl terminus β-sheet hairpin structure between amino
acids 65–75 that is homologous to an α-bungarotoxin-binding peptide [107–109]. The carboxyl
terminus region contains multiple PXXP motifs that may mediate Src homology 3 (SH3) ligand
binding. In addition, the carboxyl terminus region contains a cryptic nuclear localization sequence
(NLS) [80]. The p13 protein is mainly localized within the inner membrane of mitochondria [107,108].
However, when expressed at high levels, p13 is able to localize within the nucleus and, when
coexpressed with Tax, is directed to nuclear speckles [39]. Localization of the p13 protein within
mitochondria and the nucleus suggests that this protein may modulate effects on apoptosis and
transcriptional regulation (Figure 4).

Viruses 2011, 3 873
4.1. K+ Influx, Inner Mitochondrial Membrane Potential, and Electron Transport Chain Activity
The p13 protein was first shown to induce changes in mitochondrial morphology and distribution
[107,108,110]. Mitochondria of p13-expressing cells are clustered and have a rounded ring or
crescent-like form that differs from the typical filamentous shape and interconnected mitochondrial
network that occurs in normal cells. The rounded shape of mitochondria in p13-expressing cells is
formed by osmotic swelling in response to energy-dependent uptake of monovalent cations, such as
K+ [107,108,110]. p13 mediates this effect by altering the inner mitochondrial membrane potential
(Δψ) to change K+ permeability of these organelles. The effects of p13 on K+ permeability are
dose-dependent (Figure 4(1)). At low concentrations, p13 induces mitochondrial swelling without
causing mitochondrial depolarization or cytochrome c release which is reversed by mitochondrial
depolarization using protonophores. Cytochrome c is a component of the electron transport chain in
mitochondria and is involved in initiation of apoptosis. At higher concentrations, p13 induces
irreversible mitochondrial swelling, depolarization, and cytochrome c release. The change in
p13-mediated mitochondrial morphology is similar to, but distinct from, the changes induced by the
mitochondrial permeability transition pore (MPTP), a large nonspecific channel that regulates
cytochrome c release and apoptosis.
p13-induced K+ influx and mitochondrial membrane depolarization stimulates electron transport
and mitochondrial respiration, which increases O2 consumption [110]. Modulation of respiratory chain
activity by p13 is accompanied by increased mitochondrial reactive oxygen species (ROS) production
(discussed in Section 4.3) [110,111]. Increased ROS levels, together with mitochondrial membrane
depolarization, decrease the opening threshold of the MPTP to promote pro-apoptotic signaling.
Interestingly, at low concentrations of p13, increased electron transport chain activity dampens the
effects of p13 on Δψ by extruding H+ from the matrix [110].
4.2. Ca2+ Homeostasis
Changes in mitochondrial Δψ also regulate intracellular Ca2+ homeostasis (Figure 4(2))
[107,110,112,113]. This effect is tightly linked to the ability of p13 to induce mitochondrial K+ influx
and depolarization [113]. A p13 peptide was found to induce rapid efflux of Ca2+ from preloaded
mitochondria [107]. Though p13 reduces mitochondrial Ca2+ uptake, it does not significantly affect
overall change in cytosolic Ca2+ concentration, suggesting that p13-mediated mitochondrial
depolarization may alter Ca2+ concentration only locally [113]. By altering Ca2+ homeostasis, p13
increases the sensitivity of cells to Ca2+-mediated stimuli [112]. Increased apoptosis is observed in
p13-expressing cells upon treatment with C2 ceramide, which induces influx of Ca2+ into mitochondria
and opening of the MPTP [112]. Additionally, treatment of cells with histadine results in a rise in
cytosolic Ca2+ levels, leading to phosphorylation of CREB on serine 133 [112]. When p13 is expressed
in cells treated with histadine, there is increased nuclear accumulation of phosphorylated CREB [112].
In cells with mitochondrial defects, such as is observed in p13-expressing cells, increased CREB
phosphorylation has been shown to impair cell proliferation [114].

Viruses 2011, 3 874
4.3. ROS Production
In isolated mitochondria, p13 increases ROS production and this effect is associated with K+ influx,
mitochondrial membrane depolarization, and activation of the electron transport chain (Figure 4(3))
[110,111]. Unexpectedly, in transformed T-cells cultured using standard conditions, p13 does not
increase ROS production [111]. However, in response to glucose deprivation, p13 increases ROS
production and cell death in these cells [111]. There is a distinct gradient of ROS accumulation
between primary and transformed T-cells, with very low levels observed in resting cells, higher levels
in stimulated cells, and substantially higher levels in transformed cells [111]. In contrast to the
effects of p13 on transformed T-cells, expression of p13 in unstimulated primary T-cells induces
ROS-dependent T-cell activation and proliferation [111]. Thus, by increasing mitochondrial ROS
production, p13 mediates activation of primary resting T-cells while promoting cell death in
transformed T-cells. By modulating ROS levels in T-cells, it is possible that p13 has a role in lifelong
persistence of HTLV-1 in the host by increasing the pool of untransformed infected cells while
decreasing the number of transformed cells.
4.4. Effects on Apoptosis
Expression of p13 reduces proliferation rates of transformed cells in vitro and tumor growth in vivo.
As discussed in Section 4.1, MPTP-mediated mitochondrial swelling and altered permeability has a
key role in inducing apoptosis. Though p13 triggers similar effects as the MPTP, in addition to
inducing cristae fragmentation, it does not directly cause apoptosis or cytochrome c release [108].
Instead, p13 increases cell sensitivity to pro-apoptotic stimuli such as Fas ligand (FasL), C2 ceramide,
and glucose deprivation [111,115]. The effects of p13 on FasL-mediated apoptosis are enhanced by
overexpression of Ras and antagonized by inhibiting Ras farnesylation and subsequent activation
[115]. Upon FasL stimulation, farnesylated Ras traffics to mitochondria and directly binds Bcl-2 to
inhibit the anti-apoptotic effects of Bcl-2 [115]. Treatment of p13-expressing cells with C2 ceramide
results in increased influx of Ca2+ into mitochondria followed by opening of the MPTP, leading to cell
death [111]. During glucose deprivation, p13 promotes apoptosis by increasing ROS production in
transformed T-cells [111]. Consistent with the central role of mitochondria in energy production,
cation flux, and apoptosis, p13 is able to affect this organelle to influence cell turnover.
4.5. Nuclear Effects
In cells expressing Tax, p13 becomes ubiquitinated and is partially localized within the nucleus
(Figure 4(4)) [116]. Interestingly, Tax mediates ubiquitination of p13 though this protein contains no
lysine residues. Instead, p13 is likely ubiquitinated on serine and threonine residues and this
modification increases the stability of the protein. Within the nucleus, ubiquitinated p13 associates
with Tax to inhibit its binding to the CBP/p300 transcriptional coactivator [116]. A decrease in
Tax-CBP/p300 complex formation results in decreased Tax-mediated viral gene transcription [116].
Thus, intracellular localization of p13 may be an additional regulatory switch between viral replication
and latency.

Viruses 2011, 3 875
Figure 4. Functions of p13. In mitochondria, p13 mediates (1) K+ influx, inner mitochondrial
membrane potential, and electron transport chain activity to affect (2) Ca2+ signaling and
(3) ROS production. (4) In the presence of Tax, p13 is ubiquitinated and translocates to the
nucleus. In the nucleus, p13 inhibits Tax-CBP/p300 complex formation to decrease
transcription of cellular and viral genes.
4.6. In Vivo Animal Model
CTLs and antibodies that recognize orf-II peptides can be detected in HTLV-1-infected individuals,
suggesting that p13 may have an important role in vivo [104,117]. In an early study to examine the role
of p13 alone in vivo, a molecular clone of HTLV-1 mutated to selectively ablate p13 failed to establish
viral infection in a rabbit model [118]. However, similar to the rabbit model studies discussed in
Sections 2.5 and 3.5, it is unknown whether the mutation that ablated p13 also affected expression of
HBZ since the amino acid change that ablated the start codon for p13 in this study would also affect
the start codon for HBZ [118]. No studies have been completed that examine the role of p13 alone in
the macaque model.
5. Conclusions
HTLV-1-associated diseases have long periods of clinical latency with infected individuals having
life-long persistence of viral-infected T-cell clones. Thus, HTLV-1-infected T-cells must be able to
avoid immune recognition. As transcription of the provirus could lead to antigen presentation and
immune recognition, it is necessary for HTLV-1 to maintain low levels of virus replication. In vivo

Viruses 2011, 3 876
studies demonstrate that HTLV-1 requires orf-I and orf-II for viral persistence. Since these proteins
play critical roles in T-cell activation, MHC-I trafficking, cytokine expression, and virus replication,
they are likely crucial for HTLV-1 maintenance of low level of virus expression in vivo. Together, p12
and p8 decrease ICAM-1, ICAM-2, and MHC-I expression at the cell surface, limiting the ability of
NK cells and CTL cells to recognize infected cells. In addition, p8 inhibits proximal TCR signaling
upon TCR stimulation to prevent T-cell activation. In contrast, within the ER, p12 promotes Ca2+
release and NFAT activation in resting cells. Thus, p12 decreases the IL-2 requirement for
proliferation in the presence of suboptimal antigen presentation. Altogether, these data suggest that the
concerted expression of p12 and p8 modulate T-cell activation and antigen presentation while
promoting proliferation of resting cells, therefore providing new target cells for the virus. Orf-II
protects HTLV-1-infected cells from immune system recognition by dampening the transcriptional
effects of Tax. In the nucleus, p30 binds and prevents export of tax/rex mRNA to the cytosol for
translation and p13, upon ubiquitination, inhibits Tax-CBP/p300 complex formation. This results in a
decrease in the transcriptional activity of Tax and subsequent virus replication. In macrophages, p30
favors the production of anti-inflammatory IL-10 while decreasing the expression of pro-inflammatory
cytokines by interacting with the transcription factor PU.1. Furthermore, like orf-I, p30 and p13
promote proliferation of resting T-cells. p30 inhibits DNA repair mechanisms to favor the growth and
persistence of infected cells. p13 modulates the production of ROS and proliferation of resting cells
while promoting apoptosis of transformed cells. The understanding of how these proteins
simultaneously protect HTLV-1-infected cells from destruction and promote their expansion leads to
new questions. Where, when, and at what level these proteins are expressed in vivo still must be
elucidated. The answers to these questions will indicate whether these proteins may provide new
targets for HTLV-1 therapeutics.
Acknowledgements
This research was supported by the Intramural Research Program of the National Institute of Health,
National Cancer Institute, Center for Cancer Research.
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