Current HIV Research, 2006, 4, 131-139 131

1570-162X/06 $50.00+.00 2006 Bentham Science Publishers Ltd.

Retrovirus Translation Initiation: Issues and Hypotheses Derived from
Study of HIV-1
Alper Yilmaz
1,4,
, Cheryl Bolinger
1,4,
and Kathleen Boris-Lawrie
*,1,2,3,4,5

Center for Retrovirus Research
1
, Departments of Veterinary Biosciences
2
and Molecular Virology, Immunology &
Medical Genetics
3
, Molecular, Cellular & Developmental Biology Graduate Program
4
, Comprehensive Cancer Center
5
,
The Ohio State University, Columbus, OH, USA
Abstract: Human immunodeficiency virus type 1 (HIV-1) has a small, multifunctional genome that encodes a relatively
large and complex proteome. The virus has adopted specialized post-transcriptional control mechanisms to maximize its
coding capacity while economically maintaining the information stored in cis-acting replication sequences. The conserved
features of the 5 untranslated region of all viral transcripts suggest they are poor substrates for cap-dependent ribosome
scanning and provide a compelling rationale for internal initiation of translation. This article summarizes key
experimental results of studies that have evaluated HIV-1 translation initiation. A model is discussed in which cap-
dependent and cap-independent initiation mechanisms of HIV-1 co-exist to ensure viral protein production in the context
of 1) structured replication motifs that inhibit ribosome scanning, and 2) alterations in host translation machinery in
response to HIV-1 infection or other cellular stresses. We discuss key issues that remain to be understood and suggest
parameters to validate internal initiation activity in HIV-1 and other retroviruses.
Keywords: Retrovirus translational control, unspliced RNA, 5 untranslated region, ribosome scanning, internal ribosome entry
site.
INTRODUCTION
Retrovirus Primary Transcription Product Can Function
as Precursor mRNA, mRNA, or Viral Genomic RNA
Retroviral genomes are multifunctional RNAs that utilize
virally encoded reverse transcriptase to replicate genomic
RNA through a proviral DNA intermediate [81]. The
provirus becomes permanently integrated into the host cell
chromosome and is expressed like a cellular gene by the host
cell transcription, RNA processing, and translation
machinery. The primary retroviral transcript interacts with
the cellular RNA processing machinery to become capped
and polyadenylated. One fraction of the retroviral transcript
behaves like a typical cellular pre-mRNA in that the
spliceosome is engaged and the intron is removed. As a
consequence, it is expected that the exon junction complex is
deposited on spliced retroviral transcripts and functions as it
does on cellular mRNAs to promote a pioneer round of
translation, followed by nuclear export and finally, steady
state translation in the cytoplasm [20, 28, 33, 50, 57, 86].
Another fraction of pre-mRNA achieves nuclear export and
translational utilization independently of splicing
commitment and this activity is trans-activated by the
essential HIV Rev protein. Rev interacts with the Rev
response element (RRE) within unspliced and incompletely
spliced viral RNAs and solicits interaction with the CRM1
nuclear export receptor [35, 65]. In the cytoplasm, the
unspliced transcript plays a dual role as mRNA template for
translation and as genomic RNA that is packaged into


*Address correspondence to this author at the Ohio State University, 1925
Coffey Road, Columbus, OH 43210, USA; Tel: 614/292-1392; Fax:
614/292-6473; E-mail: boris-lawrie.1@osu.edu

These authors contributed equally to this article.

assembling virions [17]. Experiments with metabolic
inhibitors to address the relationship between HIV-1 RNA
packaging and translation have determined that unspliced
HIV-1 RNA does not segregate into separate pools for
translation and packaging, as has been identified for murine
leukemia virus [16, 43, 54]. Lack of translation is not a
prerequisite to qualify HIV-1 unspliced RNA for packaging
into progeny virions [16]. Instead, unspliced HIV-1 RNA
functions interchangeably as an mRNA template for
translation and as genomic RNA that is packaged [16]. A
similar conclusion was reached for Rous sarcoma virus
(RSV) in a study that evaluated the relationship between
translation-dependent nonsense mediated decay and RNA
packaging [48].
Barriers Posed Against Efficient Translation of All HIV-
1 Transcripts
While translation and packaging are not mutually
exclusive processes for HIV-1, the packaging signal and
other structural motifs in the 5 untranslated regions (UTRs)
have been demonstrated to inhibit ribosome scanning and
translation initiation [29, 55, 59]. The 5 UTR is the most
conserved region in HIV-1 [7] and contains several cis-
acting replication sequences, including the Tat trans-
activation response element (TAR), poly(A) signal, primer
binding site, dimerization signal, splice donor site and the
packaging signal. Some of the same structural motifs are
maintained in spliced HIV-1 transcripts. HIV-1 encodes over
30 distinct spliced transcripts as a result of alternative
splicing of the primary transcription product [66, 73]. All of
the spliced transcripts and the unspliced gag mRNA share
the same 289 nt 5 noncoding exon (Fig. 1). Because this 5
UTR contains several of the highly conserved structured
replication motifs, ribosome scanning of the spliced HIV-1
mRNAs is likewise expected to be inefficient. In addition,
132 Current HIV Research, 2006, Vol. 4, No. 2 Yilmaz et al.
ligation to various distal exons produces a collection of 5
UTRs that are ~350 to 775 nt in length and contain
degenerate Kozak consensus sequences and upstream AUG
or CUG initiator codons (Fig. 1).
CUG can be recognized as a non-AUG initiator codon
[34] and the recognition of an upstream CUG or AUG
typically decreases initiation at downstream initiator codon
[23]. The efficiency of non-AUG initiation can be modulated
by the context surrounding the codon, the secondary
structure of the transcript, and in response to changes in the
growth status of the cells. The observation that the efficiency
of non-AUG initiation in growth-regulatory mRNAs changes
in response to changes in cellular growth conditions
indicates that the scanning preinitiation complex can be
regulated to affect the recognition of such a non-AUG codon
[34].
The conservation of a relatively long 5 UTR that harbors
complex structural motifs and upstream initiator codons
implies that both the unspliced gag mRNA and the
alternatively spliced HIV-1 transcripts are poor substrates for
ribosome scanning and efficient cap-dependent translation.
Because the replication strategy of this virus family
necessitates conservation of a long and highly structured 5
UTR, the ribosome scanning and initiation of cap-dependent
translation is expected to be inefficient for retroviruses in
general.
























Fig. (1). Organization and features of the 5 untranslated regions of the major HIV-1 transcripts. Structure of the predominant
transcript for each open reading frame is shown from among the ~30 alternatively spliced transcripts that are detectable by RT-PCR [66].
Colored lines highlight sequences that comprise the various 5 UTRs. The blue line depicts the first noncoding exon, which is 289 nts in
length and is present in all HIV-1 transcripts. The black line denotes the additional 47 nts that are maintained in the unspliced transcript,
which is mRNA template for translation to Gag and Gag-Pol protein and is genomic RNA that is packaged into progeny virions. The gray
lines represent coding regions and dashed lines denote introns. AUG indicates the translation initiation codon. Also indicated is the number
of CUG and AUG codons that are present upstream of actual initiation codon. Translation to Vpu and Env is achieved from the same
transcript. The vpu AUG is subject to leaky scanning and scanning may continue until recognition of the downstream env AUG. The unused
AUG in this bicistronic transcript is labeled in parenthesis. Depiction of predicted secondary structure of replication motifs is adapted from
[7]; the motifs include the trans-activation responsive sequence; primer binding site; dimer initiation site; 5 splice site; and core RNA
packaging signal. Alternatively spliced exons, which are depicted in color, may induce variations in the folding of the upstream region,
which are not shown. Free energy predictions were calculated using Mfold [51, 87]. Deviations from Kozak consensus are indicated in
lowercase letters; underlined nts indicate those most critical for efficient cap-dependent translation initiation.
Retrovirus Translation Initiation Current HIV Research, 2006, Vol. 4, No. 2 133
HIV-1 UTRs May Mediate Alternative Initiation
Strategies
An interesting possibility is that the variant 5 UTRs
created by alternative splicing induce unique RNA
conformations that serve to affect differential modulation of
HIV-1 translation initiation. An example of this paradigm is
provided by experiments with ornithine decarboxylase
(ODC) mRNA, which have identified two alternatively
spliced ODC RNAs that use different translation initiation
strategies [69]. The ODC gene product is a rate limiting
enzyme for polyamine biosynthesis. The utilization of an
internal initiation mechanism ensures that this short-lived
enzyme (t, 10-20 min) is synthesized at a peak level during
G2/M phase. One ODC mRNA isoform contains a 273 nt 5
UTR that poses structural barriers to efficient ribosome
scanning to the downstream initiator codon. The 5 UTR of
the other isoform contains an additional 30 nt and folds into
an internal ribosome entry site (IRES) that supports internal
initiation. This IRES is efficient during G2/M, but is
inefficient during the other phases of the cell cycle [68].
These findings illuminate the possibility that unique features
of the 5 UTRs of alternatively spliced retrovirus transcripts
may choreograph alternative initiation strategies. In
particular, alternative 5 UTRs of one or more HIV-1
transcripts may contain an IRES that facilitates the synthesis
of the respective viral gene products in a cell cycle-
dependent manner.
Global Cap-Dependent Translation Initiation is
Suppressed During Cellular Stress
Conditions including virus infection, hypoxia,
progression to apoptosis and cell cycle arrest compromise
global mRNA translation [42, 76, 77, 85]. Initiation of
apoptosis triggers caspase-induced cleavage of eIF4G
scaffold protein, which produces a global decrease in cap-
dependent translation [15]. During the G2/M phases of the
cell cycle, eIF4E dephosphorylation blocks formation of the
eIF4F complex and reduces the magnitude of cap-dependent
translation [11]. IRES-containing cellular mRNAs figure
prominently under these conditions in controlling cell
growth and cell cycle progression [67, 70].
While global cellular translation is suppressed by as
much as 80% upon experimental induction of apoptosis by
treatment with etoposide [85], translation of selected
mRNAs is sustained. For example, translation of the cellular
inhibitor of apoptosis protein 1 (cIAP-1) transcript is
sustained. The cIAP-1 transcript contains an 1100 nt 5 UTR
with IRES activity that is activated during G2 arrest and
progression to apoptosis [85]. The ability to sustain or even
increase translation during G2 arrest and progression to
apoptosis is consistent with previous analysis of replication
of HIV-1 in T cells. The multi-functional HIV-1 accessory
protein viral protein R (Vpr) induces G2 arrest and drives
progression to apoptosis (reviewed in another issue of this
journal) [3]. Studies of Goh et al. [32] identified that HIV-1
transcription is increased and Gag production remains robust
during Vpr-induced G2 arrest. These observations suggest
that HIV-1 protein synthesis is not hindered by a global
reduction in cap-dependent translation. An interesting
possibility is that HIV-1 sustains translation during G2 arrest
by adopting an alternative initiation strategy such as cap-
independent internal initiation.
Thus, in addition to features of the 5 UTR that pose
barriers to ribosome scanning, another challenge to retroviral
protein synthesis would be crippling of the host cell
translation machinery as a consequence of virus-induced cell
cycle arrest or other cellular stress. Together these hurdles
stand against the generation of protein products from both
the viral unspliced pre-mRNA and the collection of
alternatively spliced transcripts. The need to sustain
translation in the face of barriers to ribosome scanning and
deleterious virus-host interactions implies that retroviruses
can adopt an alternative translation initiation strategy. The
following sections of this article discuss the major translation
initiation mechanism in eukaryotic cells in relation to an
alternative translation initiation strategy of internal ribosome
entry. The final section of the article summarizes findings
that have addressed whether or not internal initiation is a
bona fide control mechanism of importance to HIV-1
replication.
Cap Recognition and Ribosome Scanning Characterize
Translation Initiation on Typical Cellular mRNAs
Translational control regulates expression of 30% of the
eukaryotic proteome and plays a pivotal role in the cellular
response to stress and other changes in cellular growth
conditions [53]. Initiation is the rate-limiting step in protein
synthesis and the primary target for translational control
[30]. The prevailing model for translation initiation describes
the strategy used by the majority of cellular mRNAs. The
ribosome scanning model prescribes that efficiently scanned
mRNAs are monocistronic, contain a m7G(5)ppp(5)N cap
structure at their 5 termini, and have UTRs that are <100 nt
in length and are relatively unstructured [45, 46]. A
conserved sequence adjacent to the initiation codon, which is
designated the Kozak consensus (GCC (A/G)CC AUG G),
promotes efficient recognition of a particular AUG [47]. The
5 cap mediates interaction with the multicomponent eIF4F
preinitiation complex, which recruits the small ribosomal
subunit [53].
Briefly, the eIF4F complex is composed of the eIF4E cap
binding protein, the eIF4G modular scaffolding protein, and
the eIF4A RNA helicase [53]. eIF4G acts as a scaffold to
connect eIF4A and eIF3 with eIF4E that is bound to the 5
cap. eIF4A provides RNA helicase activity to remodel the 5
UTR RNA and present an open single stranded template for
the small ribosomal subunit to scan. Thus, eIF4F activates
the mRNA template for interaction with the 43S ribosome
complex, which consists of the 40S ribosomal subunit,
eIF1A, eIF3, and eIF2-GTP bound to an initiator tRNA. The
charged small ribosomal subunit scans in the 5 to 3
direction until an initiator codon embedded in a Kozak
consensus (Fig. 1) is recognized. The 60S ribosomal subunit
joins the small ribosomal subunit and the elongation of the
polypeptide chain proceeds.
Modulation of ribosome scanning is a distinguishing
feature of at least one HIV-1 transcript. As depicted in Fig.
1, Vpu and Env are translated from the same mRNA with
initiation occurring at separate AUG initiation codons.
Initiation at the downstream AUG to produce Env is reliant
on leaky scanning of the upstream vpu AUG by the 43S
ribosomal subunit [74]. When the context of the Vpu AUG is
mutated match a strong Kozak consensus and reduce
readthrough, Env translation is abolished [74]. On the other
134 Current HIV Research, 2006, Vol. 4, No. 2 Yilmaz et al.
hand, a higher level of Env translation is achieved upon
mutation of the vpu AUG [72]. Thus, an important
mechanism to achieve balanced expression of these gene
products is regulation of translation initiation by leaky
scanning.
Internal Initiation is Necessary for Translation of
Picornavirus mRNAs
An alternative to initiation by ribosome scanning was
identified initially in the Picornaviridae virus family, which
includes poliovirus and encephalomyocarditis virus (EMCV)
[41, 61]. In distinction from cap-dependent mRNA
templates, the mRNA of picornaviruses lacks the 5 m7G
cap and also contains long 5 UTRs of up to ~750 nt in
length that form stable secondary structures and contain
oligopyrimidine tracts and multiple cryptic AUG or CUG
codons positioned upstream of the authentic initiation codon
[27]. As described above, these features are deleterious to
initiation by a ribosome scanning mechanism. Here, internal
recognition of the in-frame start codon at an IRES is
responsible for successful recruitment of the small ribosome
subunit [27]. Members of the Picornaviridae are completely
dependent on the IRES to initiate viral protein synthesis [19,
41, 61]. IRES is a structural motif that interacts with IRES
transacting factors (ITAF) such as Lupus antigen (La) [44,
52] and polyprimidine tract binding protein [12, 36] and is
recognized by a modified preinitiation complex [62]. The
IRES promotes recruitment of the charged small ribosomal
subunit independently of cap-binding [37].
Global Translation Shutdown is Induced by
Picornavirus, but not HIV-1 Infection
Picornaviruses also have adapted convergent mechanisms
that drastically disrupt the host capacity for cap-dependent
initiation, which induce preferential IRES-mediated
translation [6]. Poliovirus 2A protease causes rapid and
complete shutdown of cap-dependent translation by
proteolytic cleavage of eIF4G, while cleaved fragments of
eIF4G1 remain competent for IRES-mediated translation
initiation [2, 56]. EMCV infection does not induce cleavage
of eIF4G but induces dephosphorylation of eIF4E-BP,
resulting in complex formation between eIF4E and eIF4E-
binding protein, which sequesters eIF4E from interaction
with capped mRNA and eIF4G [5]. This suppressive activity
can be mimicked by treatment with rapamycin, a drug that
suppresses cap-dependent translation by shutting down the
mTOR pathway [5, 31, 60]. Under conditions of normal
cellular growth, mTOR phosphorylates eIF4E-BP, thereby
releasing eIF4E for interaction with capped mRNAs, and
providing a mechanism for the cell to bolster cap-dependent
translation.
Agy and Katze investigated the possibility that HIV-1
disrupts host cell translation [1]. Using metabolic labeling of
HIV-1 infected T cells, they showed that HIV-1 infection
correlated with a reduction in global protein synthesis. In
distinction from poliovirus, this activity correlated with a
parallel reduction in global RNA level. A collection of
gradient analyses of HIV-1 infected T cells determined that
HIV-1 infection correlates with small, but reproducible
differences in ribosome profiles [16]. These results

eliminated the possibility that HIV-1 induces the rapid and
dramatic global shutdown of host cell translation that is
characteristic of poliovirus infection. The significance of the
small change in ribosomal profile in response to HIV-1
infection remains to be determined.
Internal Initiation is Also Used by a Small but Important
Minority of Cellular Transcripts
Initially, the capacity for internal initiation was
postulated to be a strategy by which picornaviruses and
possibly other viruses monopolize the cellular translation
machinery. This view has been challenged by the discovery
that selected cellular mRNAs contain an IRES [reviewed in
37]. Furthermore, in at least some cases, the capacity for
internal initiation on cellular mRNAs coexists with the
capacity for cap-dependent initiation and may provide a
reliable mechanism to sustain de novo synthesis of critical
proteins during conditions that compromise global cellular
translation.
Studies of fibroblast growth factor 2 (FGF2) mRNA
showed that cap-dependent and cap-independent translation
initiation can occur from the same 5 UTR [10]. The
respective initiation mechanisms utilize separate start codons
and produce alternative protein isoforms. Study of human
FGF2 has mapped five initiation codons, four CUG and one
AUG, within an alternatively translated region. The 5-
proximal CUG codon is recognized in a cap-dependent
manner whereas the four distal initiation codons are
recognized in the context of an IRES [10, 82]. Alternative
internal initiation is stimulated by interaction with hnRNP
A1, which functions as an ITAF [9]. When hnRNP A1 is
depleted by RNA interference, internal initiation from the
four distal initiation codons is eliminated.
The Bcl-2 associated athanogene-1 (BAG-1) 5 UTR
provides another example of a 5 UTR that can support
translation initiation by both cap-dependent and cap-
independent mechanisms. In this case, IRES-mediated
internal translation is stimulated by the ITAF polypyrimidine
tract binding protein and poly r(C) binding protein [63].
These proteins bind and induce secondary structure changes
correlated with ribosome binding [64].
Coordinate Utilization of Cap-Dependent and Cap-
Independent Initiation is a Versatile Translation Strategy
High level expression of BAG-1 and selected other
transcripts that can utilize internal initiation (such as c-Myc
[22, 78, 79], Apaf-1 [21], Bcl-2 [75], XIAP [40], DAP5 [38])
is expected to be detrimental under normal conditions of cell
growth. In at least some of these transcripts, their IRES may
have evolved in response to selective pressure to maintain
low levels of translation during cell growth, while sustaining
or, in some cases, potently increasing protein production
upon cellular stress and cycle arrest. A similar ability to
respond to changes in cell growth conditions may be
advantageous for HIV-1 in infected T cells, where low
immunogenicity is favorable. The ability to coordinately
utilize cap-dependent and cap-independent initiation could
provide HIV-1 an advantageous strategy to sustain low
levels of translation and consequentially virus production

Retrovirus Translation Initiation Current HIV Research, 2006, Vol. 4, No. 2 135
during cell growth, and sustain or even potently increase
translation during G2 arrest.
STUDIES OF IRES-LIKE ACTIVITY IN HIV-1
A central goal of this article is to explore whether or not
internal initiation has been shown to be a bona fide control
mechanism of importance in HIV-1 replication. The
following section summarizes experimental analyses
undertaken to address internal initiation in HIV-1.
Evidence Against Internal Initiation on HIV-1 Unspliced
RNA
As discussed above, compelling rationale exists in
support of a role for internal ribosome entry in HIV-1
translational control. However, drawing such a conclusion
from the experimental analyses has been less
straightforward. At least three studies concluded that the
HIV-1 unspliced gag-pol RNA does not contain an IRES
[29, 55, 59]. At least two other studies have identified IRES-
like sequences in the unspliced transcript, either in the 5
UTR [13] or within the gag coding sequence [14]. The
experimental approaches have included bicistronic reporter
assays in transfected cells and reporter assays in extracts
prepared from rabbit reticulocytes. To date, the analysis has
not been extended to a study of HIV-1 provirus in infected T
cells.
The bicistronic reporter assay measures protein synthesis
from polycistronic reporter RNA that contains an intergenic
sequence composed of the candidate IRES. The 5 cistron
provides a measure of cap-dependent translation, while the
3 cistron provides a measure of cap-independent translation
that would be mediated, in response to the candidate IRES,
by internal ribosome entry. Potential alternative explanations
for activity from the 3 cistron are internal transcription from
a cryptic promoter and alternative splicing that ligates the 3
cistron in-frame with 5 cistron sequences to create a cap-
dependent form of the 3 open reading frame [84].
Miele et al. tested a bicistronic reporter plasmid that
expresses firefly luciferase in the 5 cistron and
chloramphenicol acetyltransferase (cat) in the 3 cistron
[14]. The 5 UTR of HIV-1
pSVC21
was placed in the
intergenic region. Protein extracts from transfected HeLa or
COS cells were standardized with respect to firefly
Luciferase units and evaluated for CAT activity. The
positive control IRES from poliovirus yielded 21-23%
acetylation. By comparison, the HIV-1
pSVC21
5 UTR
produced CAT activity equivalent to the negative control of
an unrelated spacer sequence (1.3% acetylation). In addition,
Miele et al. presented results of in vitro translation assays in
rabbit reticulocyte lysate showing that introduction of the
HIV-1
pSVC21
5 UTR to gag reporter RNA reduced protein
synthesis by 75% in comparison to a UTR deletion mutant.
Together, the analyses showed that the HIV-1
pSVC21
5 UTR
is not a translation enhancer and instead acts to reduce
translation, probably by inhibiting ribosome scanning.
Buck et al. evaluated a bicistronic reporter plasmid in
which HIV-1 env was placed in the 5 cistron, HIV-1
LAI
gag
was placed in the 3 cistron, and segments of the HIV-1
LAI
5
UTR were evaluated in the intergenic region [14].
Transfected COS-7 cells were analyzed for Env and Gag
protein production by immunoblot or ELISA. HIV-1 Env
production was barely detectable, whereas significant Gag
protein was produced. Since the env coding region contains
multiple AUGs and is predicted to form strong secondary
structure, this sequence was interpreted to inhibit read-
through activity that would yield Gag protein. Consistent
with the results of Miele et al. [55], partial or complete
deletion of the 5 UTR increased Gag production by 2-fold
over the parental reporter containing the intact 5 UTR.
Because the 5 UTR did not confer Gag production, the
authors concluded that the gag 5 UTR is dispensable for
Gag production and that the gag 5 UTR does not contain
IRES activity.
Evidence in Support of Internal Initiation on HIV-1
Unspliced RNA
Buck et al. also evaluated a bicistronic reporter plasmid
in which CAT was placed in the 5 cistron, HIV-1
LAI
gag
was placed in the 3 cistron, and a 77 bp fragment of the
HIV-1
LAI
5 UTR was placed in the intergenic region [14].
Infection with poliovirus was used to suppress cap-
dependent translation in transfected 293T cells over a period
of six hours. Protein synthesis was evaluated by
immunoprecipitation with antibodies against CAT and HIV-
1. CAT protein synthesis was reduced by a factor of six,
which is consistent with suppression of cap-dependent
translation by poliovirus. By contrast, Gag protein synthesis
was not inhibited, and this trend mimicked that of the EMCV
IRES-cat control. Northern blot analysis eliminated the
possibility that gag mRNA translation initiated from cap-
dependent alternative transcripts generated by aberrant
splicing or by cryptic promoter activity. The authors
conclude that since synthesis is sustained of Gag Pr55
protein from bicistronic reporter RNA during shutdown of
cap-dependent translation by poliovirus, IRES activity is
conferred by sequences within the gag gene.
Brasey et al. used bicistronic renilla luciferase and firefly
luciferase RNA to systematically evaluate sixteen segments
of the HIV
NL4-3
5 UTR and gag open reading frame for
IRES activity. In contrast to the results of Miele et al. and
Buck et al., this analysis identified IRES activity in the HIV
5 UTR in HeLa cells. Results of dual Luciferase assays
indicated that reporters containing the HIV
NL4-3
5 UTR
produced firefly Luciferase activity at a level similar in
magnitude to the poliovirus IRES positive control. When the
5 UTR was extended to include gag sequences, firefly
Luciferase activity was reduced. The results in this
bicistronic reporter system conflict with the conclusion by
Buck et al. that gag coding sequences support IRES activity.
A possible explanation is differences in the structure of the
bicistronic reporter constructs.
Brasey et al. used nocodazole to induce cellular G2 arrest
and investigate the possibility that HIV-1 IRES activity is
active during cell cycle arrest and the consequential
suppression of cap-dependent initiation [13]. HeLa cells
were transduced with a bicistronic murine leukemia virus
(MLV)-based retroviral vector that contained the HIV
NL4-3
5
UTR in the intergenic region between 5 placental alkaline
phosphatase (plap) and 3 neomycin phosphotransferase
(neo) reporter cistrons. Metabolic labeling showed that
nocodazole treatment suppressed total cellular protein
synthesis to 47% of the control. Immunoblot results
136 Current HIV Research, 2006, Vol. 4, No. 2 Yilmaz et al.
determined that Neo protein synthesis was not reduced but
rather was maintained at a level of 168% of the untreated
control. The observation in the context of this bicistronic
reporter construct that translation is sustained during
nocodazole-induced G2 arrest supports the conclusion that
sequences of the HIV-1 5 UTR support cap-dependent
translation initiation.
Indirect evidence supporting the presence of an IRES in
the HIV-1 UTR is derived from the observation of
interaction between La autoantigen and the HIV-1 5 UTR
[18]. La has been shown to be an ITAF that is necessary for
EMCV IRES-mediated translation [44]. Results of mobility
shift assay and detection of HIV-1 TAR RNA in
immunoprecipitates from HIV-1 infected lymphocytes using
anti-La serum showed that La binds the 5 UTR by
recognizing TAR secondary structure in vitro and in vivo
[18]. Moreover, this interaction correlated with increased
translation from HIV-1 TAR-containing CAT mRNA in
rabbit reticulocyte lysates [80]. Genetic analysis will be an
informative approach to further explore the possibility that
La is an ITAF that modulates HIV-1 IRES activity.
Another study used rapamycin to interrogate the 5 UTR
of the related primate retrovirus, SIV
mac
for evidence of
internal initiation [58]. These experiments utilized an MLV-
based retroviral vector that expressed plap in a 5 cistron and
neo in the 3 cistron with the intergenic region containing the
SIV 5 UTR. Rather than transient transfection assays, NIH-
3T3 cells were transduced with the retroviral bicistronic
reporter vectors. The transductants were subjected to long-
term selection with G418 for exclusive selection of cells
expressing the 3 cistron of the bicistronic reporter RNA.
Then G418-resistant clones were treated with rapamycin to
disrupt cap-dependent translation by inactivation of the
mTOR pathway. Rapamycin treatment for six hours
decreased Plap activity by 43% relative to an untreated
control. Under these conditions, Neo activity increased two-
fold. These results are consistent with internal initiation on
the SIV 5 UTR within these G418-selected transduced cells.
STUDIES OF IRES-LIKE ACTIVITY IN OTHER
RETROVIRUSES
Capacity for internal initiation in other retroviruses is
also being evaluated in studies with bicistronic reporter
plasmids. Sequences from HIV-2, human T lymphotropic
virus type 1 (HTLV-1), reticuloendotheliosis virus strain A
(RevA), RSV, and MLV have been shown to be capable of
directing IRES activity in reporter mRNA [4, 8, 13, 24, 25,
39, 49, 58, 83]. One example of these studies tested the 5
UTRs of RSV and vSRC for IRES activity in the context of
bicistronic neomycin-lacZ reporter plasmid [25]. Significant
translation of lacZ was produced in response to the RSV and
vSRC containing reporters even when a stable (-50
Kcal/mol) hairpin was inserted upstream of the 5cistron to
inhibit ribosome scanning and control for reinitiation. A
separate study looked for MLV IRES activity in NIH3T3
cells permanently expressing bicistronic reporter RNA
containing the 5 leader of MLV envelope between placental
alkaline phosphatase (plap) and neomycin, respectively.
After treatment with rapamycin to inhibit cap-dependent
translation, plap activity decreased by 26% while neomycin
expression increased by 316% when compared to untreated
cells [24]. A similar approach was used to identify an IRES
in the 5 UTR of RevA [49]. The RevA IRES was recently
shown to facilitate translation initiation in mouse neural
precursors and to be capable of maintaining activity after cell
differentiation, indicating that ubiquitous ITAF are utilized.
[26]. Overall, the studies of bicistronic reporter plasmids
with sequences from several divergent retroviruses identify
small, but positive stimulation of internal initiation. A bona
fide role for internal initiation in translation of their
respective natural viral transcripts remains to be determined.
Consideration of Unified Parameters to Define Positive
IRES Activity
As enumerated by van Eden et al., bicistronic reporter
gene assays performed in combination with structural and
quantitative RNA analysis have been a productive approach
to identify candidate viral and cellular IRESes [84]. For the
retroviral literature, effective comparison of the magnitude
of IRES activity identified in the various bicistronic reporter
assays has been difficult. Two challenges to effective
comparison of the results of studies have been differences in
the definition of the baseline activity and differences in the
definition of positive IRES activity.
RNA transfection of bicistronic reporter transcripts is an
alternative approach to plasmid-based transfection assays.
This RNA-based approach overcomes the potential pitfall
that protein production is mediated by alternative transcripts
generated by cryptic promoter activity or aberrant splicing,
rather than internal ribosome entry on the bicistronic reporter
RNA [84]. Experiments with viral or chemical treatment to
inhibit cap-dependent translation have provided a
complementary aspect to the bicistronic reporter assay [13,
14] and could be further utilized for analysis of natural
mRNA templates expressed from authentic provirus.
Furthermore, this approach is useful to validate authentic
cap-independent translation, which is an expected
characteristic of an mRNA that contains a typical IRES. This
approach may also be useful to address the possibility of
activity conferred by a cap-dependent ribosome shunt [71].
The inconsistencies between results of the bicistronic
assays to investigate IRES activity in HIV-1 sequences may
also be attributed to: 1) the caveat that the bicistronic
reporter system evaluates HIV-1 sequences in an artificial
context that may not recapitulate the natural HIV-1 RNA
secondary or tertiary structure; 2) fibroblastic cell lines were
used rather than CD4+ human T-lymphocytes, a natural
target for HIV-1. These cell lines are expected to present a
unique set of ITAFs, each of which may or may not be
important for the activity of natural IRESes present in the
HIV-1 genome. The inconsistencies exhibited between the
results strongly warrant testing in CD4+T-lymphocytes; 3)
possible interplay between cap-dependent translation and
IRES-mediated translation may affect internal entry in the
natural HIV-1 RNA context, which is not recapitulated in the
bicistronic reporter system. A challenge is to go beyond the
artificial bicistronic approach and evaluate translation
initiation in the context of the HIV-1 provirus. Evaluation of
natural HIV-1 mRNAs expressed from provirus in primary
lymphocytes is necessary to validate that internal initiation is
a bona fide control mechanism of importance during HIV-1
replication.
Retrovirus Translation Initiation Current HIV Research, 2006, Vol. 4, No. 2 137
Clues Supporting Coordinate Cap-Dependent and Cap-
Independent Initiation on HIV-1 mRNA
Another challenge is to address the possibility that cap-
dependent and cap-independent translation mechanisms in
HIV-1 function in a coordinate manner. The existence of
redundant initiation mechanisms is supported by the
observation that HIV-1 mRNA is subject to cap-dependent
ribosome scanning and that HIV-1 sequences exhibit IRES
activity in at least some bicistronic reporter assays. What
criteria are pertinent to determine which mechanism is
dominant and what conditions favor use of the cap-
dependent versus cap-independent mechanism? Cap-
dependent translation initiation is used by most cellular
RNAs and is expected to be a more efficient mechanism then
internal initiation, therefore, yielding more protein product.
On the other hand, constitutive high level synthesis of viral
proteins may be detrimental to the growth and viability of
the host cell and increase immunogenicity, which may be
deleterious to the progression of HIV-1 replication. Internal
initiation enabled by a regulatable IRES would be a versatile
strategy to fine-tune viral protein production in response to
progression of virus infection and/or changes in the cellular
environment. Early in HIV-1 infection, a cap-dependent
mechanism may be predominant and sustain a level of viral
protein synthesis that is not deleterious to the cell. In later
stages of infection, or in response to increased stress in the
cellular environment, the cap-dependent mechanism may be
replaced by the internal initiation mechanism. This
alternation between initiation strategies would provide a
mechanism for HIV-1 to sustain translation during the G2
arrest that is induced by Vpr. Identifying which translation
initiation mechanism(s) is utilized by HIV-1 during
sequential phases of host cell infection may reveal new
targets for antiviral therapy. Identification of ITAFs that
modulate internal initiation from various HIV-1 mRNAs will
provide new insight into how this virus modulates viral
protein under conditions of cellular distress and against the
odds.
ACKNOWLEDGEMENTS
We are grateful to Tim Vogt for figure preparation and
Dr. Kate Hayes for editing and Dr. Ihab Younis, Nicole
Placek and Shuiming Qian for critical comments on the
manuscript. This work was supported by National Institutes
of Health National Cancer Institute P01 CA100730 and P30
CA16058.
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Received: September 15, 2005 Revised: December 9, 2005 Accepted: December 13, 2005