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Retroviruses exploit nuclear trafficking machinery at several distinct stages in their replication cycles. In this review, we will focus primarily on nucleocytoplasmic trafficking events that occur after the completion of reverse transcription and proviral integration. First, we will discuss nuclear export of unspliced viral RNA transcripts, which serves two essential roles: as the mRNA template for the translation of viral structural proteins and as the genome for encapsidation into virions. These full-length viral RNAs must overcome the cell's quality control measures to leave the nucleus by co-opting host factors or encoding viral proteins to mediate nuclear export of unspliced viral RNAs. Next, we will summarize the most recent findings on the mechanisms of Gag nuclear trafficking and discuss potential roles for nuclear localization of Gag proteins in retrovirus replication.
Model of retrovirus replication. Retroviral infection is initiated with binding of the viral Env protein to a cell surface receptor and fusion of the viral envelope with the cellular membrane (step 1). The viral RNA genome is reverse transcribed from RNA to DNA (step 2) to form the provirus, which is stably integrated into the genome of the host cell (step 3). Viral RNA is transcribed by the host polymerase II, and a portion of the RNA is spliced to direct the translation of the Env glycoprotein and other viral proteins (step 4). A portion of the viral RNA remains unspliced and is exported from the nucleus by the host factor TAP/NXF1 (e.g., simple retroviruses MPMV and RSV) or virally encoded Rev-like proteins (e.g., complex retroviruses like HIV-1 and HTLV-I) (step 5) to serve as a template for Gag and Gag-Pol translation (step 6). The Gag proteins of some retroviruses traffic through the nucleus during assembly (step 7). It has been postulated that the nuclear population of Gag (denoted by “?”) might select genomic RNA (gRNA) a nd transport it into the cytoplasm for packaging. Alternatively, selection of genomic RNA may occur in the cytoplasm (step 8). In either case, the Gag-gRNA complex is transported to the plasma membrane (step 9) where additional Gag molecules bind the viral RNP to complete assembly of the immature virus particle, which buds from the plasma membrane (step 10). The steps in replication that are covered in this review are indicated in bold. Figure modified from [22] and used with the author’s permission.
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Viruses 2013, 5, 2767-2795; doi:10.3390/v5112767
ISSN 1999-4915
Nuclear Trafficking of Retroviral RNAs and Gag Proteins
during Late Steps of Replication
Matthew S. Stake 1, Darrin V. Bann 1, Rebecca J. Kaddis 1 and Leslie J. Parent 1,2,*
1 Division of Infectious Diseases and Epidemiology, Department of Medicine, Penn State College of
Medicine, 500 University Drive, Hershey, PA 17033, USA; E-Mails: (M.S.S.); (D.V.B.); (R.J.K.); (L.J.P)
2 Department of Microbiology & Immunology, Penn State College of Medicine, 500 University
Drive, Hershey, PA 17033, USA
* Author to whom correspondence should be addressed; E-Mail:;
Tel.: +1-717-531-3997; Fax: +1-717-531-4633.
Received: 12 October 2013; in revised form: 31 October 2013 / Accepted: 12 November 2013 /
Published: 18 November 2013
Abstract: Retroviruses exploit nuclear trafficking machinery at several distinct stages in
their replication cycles. In this review, we will focus primarily on nucleocytoplasmic
trafficking events that occur after the completion of reverse transcription and proviral
integration. First, we will discuss nuclear export of unspliced viral RNA transcripts, which
serves two essential roles: as the mRNA template for the translation of viral structural
proteins and as the genome for encapsidation into virions. These full-length viral RNAs
must overcome the cell’s quality control measures to leave the nucleus by co-opting host
factors or encoding viral proteins to mediate nuclear export of unspliced viral RNAs. Next,
we will summarize the most recent findings on the mechanisms of Gag nuclear trafficking
and discuss potential roles for nuclear localization of Gag proteins in retrovirus replication.
Keywords: nuclear entry; nuclear export; nucleocytoplasmic trafficking; virus assembly;
viral RNA packaging; retroviral Gag protein; HIV-1 Rev protein; Rous sarcoma virus;
Viruses 2013, 5 2768
1. Introduction: Nuclear Trafficking Events in Retrovirus Replication
Retroviruses interact with nuclear trafficking machinery during several different phases of their
replication cycles (Figure 1). Retrovirus replication has been divided into “early” and “late” stages,
with early events extending from virus entry through integration and late stages encompassing
expression of viral RNA from the provirus through virus assembly and budding of immature virus
particles from the host cell. During early infection, all retroviruses must gain access to the host
chromatin for the provirus to integrate. Most retroviruses depend on mitosis and breakdown of the
nuclear envelope to undergo integration. However, lentiviruses like human immunodeficiency virus
type 1 (HIV-1) infect non-dividing cells, having developed strategies to enter the nucleus by passing
through intact nuclear pores (reviewed in [1-3]). Although several viral factors, including HIV-1 MA
(matrix), IN (integrase), Vpr, and the reverse-transcribed proviral DNA have been implicated in
nuclear entry of the pre-integration complex (PIC), more recent data indicate that the CA (capsid)
protein, nuclear import factor transportin-3 (TNPO3), and the nucleoporin Nup358 are important
determinants [4-15]. Thus, retroviruses have evolved different strategies to promote proviral
integration through complex interactions between host and viral factors. Although these early events
are essential to establish retroviral infection, this review will focus primarily on interactions of
retroviruses with nuclear transport machinery following integration.
Once integration is completed, viral RNA is synthesized by the cellular RNA polymerase II.
Retroviral transcripts are co-transcriptionally modified with the addition of a 5’ cap and 3’ poly(A)
tail, like cellular mRNAs. A fraction of the viral RNA is spliced and exported out of the nucleus to
serve as mRNA for translation into viral proteins. The remainder of the viral RNA remains unspliced
and must be transported from the nucleus into the cytoplasm where it serves two functions: (i) as the
template for translation of the viral structural proteins, Gag and Gag-Pol; and (ii) as the viral genome,
which is packaged into virus particles. Because export of unspliced and incompletely spliced cellular
RNAs is prevented by host machinery to prevent translation of abnormal proteins (reviewed in [17]),
retroviruses must circumvent this cellular blockade to export their full-length RNA molecules
(reviewed in [16,18]). Prior translation of the unspliced viral RNA is not a prerequisite for genome
encapsidation, as genomic RNA can be packaged in trans (reviewed in [16]). The genomic RNA
forms a noncovalent dimer and is encapsidated through an interaction between the psi () packaging
sequence near the 5’ end of the genome and the NC (nucleocapsid) domain of the Gag protein.
The Gag protein, which directs the assembly and budding of virus particles from the plasma
membrane, is localized primarily in the cytoplasm of infected cells. However, the Gag proteins of
Rous sarcoma virus (RSV), feline immunodeficiency virus (FIV), mouse mammary tumor virus
(MMTV), prototype foamy virus (PFV), murine leukemia virus (MLV), Mason-Pfizer monkey virus
(MPMV), HIV-1, and several retrotransposons undergo nuclear localization under certain conditions
(Table 1). The RSV, FIV, and PFV Gag proteins utilize the cellular CRM1 protein for nuclear export
[19-21], but the host importins involved in nuclear import of Gag have only been defined for RSV. In
this review, we will focus on nuclear transport events associated with the nucleocytoplasmic
trafficking of unspliced retroviral RNAs and Gag proteins and their roles in virion assembly.
Viruses 2013, 5 2769
Figure 1. Model of retrovirus replication. Retroviral infection is initiated with binding of
the viral Env protein to a cell surface receptor and fusion of the viral envelope with the
cellular membrane (step 1). The viral RNA genome is reverse transcribed from RNA to
DNA (step 2) to form the provirus, which is stably integrated into the genome of the host
cell (step 3). Viral RNA is transcribed by the host polymerase II, and a portion of the RNA
is spliced to direct the translation of the Env glycoprotein and other viral proteins (step 4).
A portion of the viral RNA remains unspliced and is exported from the nucleus by the host
factor TAP/NXF1 (e.g., simple retroviruses MPMV and RSV) or virally encoded Rev-like
proteins (e.g., complex retroviruses like HIV-1 and HTLV-I) (step 5) to serve as a template
for Gag and Gag-Pol translation (step 6). The Gag proteins of some retroviruses traffic
through the nucleus during assembly (step 7). It has been postulated that the nuclear
population of Gag (denoted by “?”) might select genomic RNA (gRNA) and transport it
into the cytoplasm for packaging. Alternatively, selection of genomic RNA may occur in
the cytoplasm (step 8). In either case, the Gag-gRNA complex is transported to the plasma
membrane (step 9) where additional Gag molecules bind the viral RNP to complete
assembly of the immature virus particle, which buds from the plasma membrane (step 10).
The steps in replication that are covered in this review are indicated in bold. Figure
modified from [22] and used with the author’s permission.
Viruses 2013, 5 2770
Table 1. Retroviral proteins that undergo nuclear trafficking*
Retroviral Protein
Localization of the Population
Associated with the Nuclear
Nuclear Localization
Nuclear Export
Nucleus [20, 23]
Punctate Nuclear Foci [23, 24]
Nucleoli [24]
Imp11 (MA domain) [25, 26]
TNPO3 (MA Domain) [26]
Importin α/β (NC Domain) [25,
CRM1 [20,25]
Nucleoli [24]
Importin α/β [25, 26]
RSV RT, β subunit
Nucleus [27]
Nucleus [28]
Shares Import Pathway with
Histone H1 [29]
Nucleoli [30]
Unknown [30]; Nucleolar
Localization Increased with
RPL9 Overexpression [30]
No Identified NES
Nucleoli [24, 30]
Nucleoli [31]
Retrotranslocation from
Endoplasmic Reticulum to
Nucleus [32]
CRM1 [31]
Nuclear Pore Complex; Low
Levels in Nucleus [33, 34]
Unknown; Nuclear localization
Increased with Ubc9
Overexpression [34]
CRM1 [35]
Nucleus and Nucleoli [36, 37]
Rej Function Depends on
CRM1 [37]
Nucleus [38]
No Identified NES
Nucleus [24, 39]
Nucleoli [24, 39]
MLV p12
Mitotic Chromatin [40, 41]
Nucleus and Nucleoli [39]
Unknown; Interacts with
Brd4 in Nucleus [42]
Nucleus and Nucleoli [43-45]
Punctate Nuclear and Nucleolar
Foci [43]
Importin β [46]
CRM1 [47,48]
Punctate Nuclear Foci [49]
Nuclear Pore Complex [49]
CRM1 [49]
HIV-1 Gag
Nucleolar [24]
Not CRM1 [19,35,50]
Nucleus [14, 51-55]
Importin α3 [51]
Importin α [14, 52]
Importin 7 [56]
Nucleoli [24]
Nucleus [57, 58]
HIV-1 Rev
Nucleus andNnucleoli [44, 59]
Nuclear Foci [59]
HIV-1 Transcription Sites [60]
Importin β [61, 62]
Transportin, Importin 5, and
Importin 7 [63]
CRM1 [47,64]
Viruses 2013, 5 2771
Table 1. Cont.
Retroviral Protein
Localization of the Population
Associated with the Nuclear
Nuclear Import Mechanism
Nuclear Export Factor
HIV-1 Vpr
Nucleus [65, 66]
Nuclear Pore Complex [6, 66]
Importin α [66]
Interacts Directly with Nuclear
Pore Complex [67, 68]
CRM1 [65]
HIV-2 Rev
Nucleoli [69]
HIV-2 Vpx
Nucleoplasm [70, 71]
No Identified NES
Nucleus [19], Nucleoli [19], and
Nuclear Envelope [72]
CRM1 [19]
Nucleolus [73]
CRM1 [74]
Nucleus [74]
CRM1 [74]
Nucleus and
Nucleoli [75, 76]
Importin [77]
CRM1 [77]
Nucleoli [78]
Nucleoli [79, 80]
Nucleus [21, 81]
Punctate Nuclear Foci [82, 83]
Mitotic Chromatin [82]
Binds to H2A/H2B on Mitotic
Chromatin [82, 83]
CRM1 [21]
* Note: Transcriptional activators related to HIV-1 Tat were not included in the table.
2. Nuclear Export of Unspliced and Incompletely Spliced RNAs of Complex Retroviruses
Productive retroviral infection requires unspliced viral transcripts to be transported into the
cytoplasm where they are translated into the essential viral proteins Gag and Gag-Pol. To circumvent
intrinsic cellular blockades that prevent the export of incompletely spliced RNAs from the nucleus,
complex retroviruses encode trans-acting viral proteins that export their intron-containing viral RNAs
from the nucleus. HIV-1 Rev was the first member of this family to be discovered; however, Rev-like
proteins have been described in the Lentivirus [e.g., Rev proteins of human immunodeficiency virus
type-2 (HIV-2), simian immunodeficiency virus (SIV), FIV, equine infectious anemia virus (EIAV),
bovine immunodeficiency virus (BIV), Maedi-visna virus (MVV) and caprine encephalitis-anemia
virus, CAEV)] [73,84-93], Deltaretrovirus [(e.g., Rex proteins of human T cell leukemia virus type-I
(HTLV-I) and bovine leukemia virus (BLV)], and Betaretrovirus [e.g., Rem protein of MMTV and
Rej protein of Jaagsiekte sheep retrovirus (JSRV)] genera [31,36,37,77,94-96]. Rev-like proteins
localize to the nucleus and nucleolus through interactions with a variety of import factors (see ( 1),
and they contain CRM1-dependent nuclear export signals (NESs) [45,47,77,97-100].
HIV-1 Rev recognizes and binds to the highly structured cis-acting Rev-responsive element (RRE)
in HIV-1 RNAs [101-104] and undergoes multimerization, which is important for its export function
(reviewed in [105]). Multimerization of Rev was demonstrated within the nucleolus in living cells,
suggesting that the nucleolus may be the site of Rev multimer formation [106]. However, the relevance
of these experiments is somewhat limited because they were not performed in the context of HIV-1
infection, nor was the RRE sequence expressed in the cells. Attempts to define whether nucleolar
localization of Rev is important for binding to RRE-containing RNAs have been difficult because the
Rev RNA binding domain overlaps with the nuclear/nucleolar localization signal. However, HIV-1
Viruses 2013, 5 2772
unspliced RNA appears to undergo nucleolar trafficking based on the finding that the RNA is cleaved
by ribozymes artificially targeted to the nucleolus [107] and small nucleolar RNAs engineered to
contain the RRE are exported into the cytoplasm by Rev [108,109]. Together, these data provide
evidence that Rev and the RRE-containing viral transcripts both traffic through the nucleolus, but there
is no definitive evidence that nucleolar trafficking of the Rev-RRE complex is essential for nuclear
export of HIV-1 RNA during natural virus infection.
In the nucleoplasm, Rev co-localizes with the SR-domain splicing factor SC-35 in nuclear
speckles [59,110], intrachromatin granule clusters enriched in mRNA splicing factors and snRNPs
located adjacent to sites of active transcription [111-115]. The association of Rev with splicing factors
in speckles suggests that Rev binds to the RRE co-transcriptionally, just as splicing factors bind to
cellular transcripts as they are synthesized [116]. In support of this idea, previous studies demonstrated
that the Rev-RRE interaction is abrogated by transcription inhibitors [117]. More recently, it was
shown that Rev co-localizes with HIV-1 RNA at transcription sites, providing strong evidence that
Rev binds RRE-containing transcripts co-transcriptionally [60].
HIV-1 Rev mediates export of unspliced viral RNAs through an interaction between the NES of
Rev and the CRM1/RanGTP export complex. However, many other cellular proteins also interact with
Rev, indicating that nuclear export of unspliced HIV-1 RNA depends upon a complex network of
interactions [60,62,105,118-127]. For example, several RNA helicases interact with Rev, including
DDX1 and DDX3, which help to maintain Rev localization in the nucleolus and nucleus [124,126-
128]. In addition, the nuclear matrix-associated protein Matrin 3 binds HIV-1 RNA co-
transcriptionally and facilitates Rev-mediated nuclear export of unspliced RNA [60,129]. Although the
precise mechanism by which Matrin 3 facilitates HIV-1 RNA export has not been elucidated, Matrin 3
increases the stability and expression of cellular mRNAs, suggesting that it may have a similar effect
on HIV-1 RNA [130]. The finding that Matrin 3 interacts with HIV-1 RNA also creates an intriguing
connection between HIV-1 RNA export and the nuclear matrix, which not only provides structural
support to the nucleus [131] but also associates with areas of euchromatin involved in ongoing
transcription [132]. Recent evidence also suggests that components of the nuclear matrix called nuclear
regulatory networks bind genomic DNA and form a tubular pathway leading to nuclear pore
complexes for nuclear export of transcripts and proteins [133,134]. Therefore, it is intriguing to
postulate that Matrin 3 bridges the interaction between Rev and active HIV-1 RNA transcription sites
[60,129,135,136], recruiting the CRM1 nuclear export machinery associated with nuclear regulatory
networks to transport viral ribonucleoprotein complexes (RNPs) through the nuclear pore and into the
3. The Role of cis-Acting Sequences in RNA Export of Simple Retroviruses
In contrast to complex retroviruses that encode trans-acting factors to facilitate nuclear export of
unspliced RNA, simple retroviruses have evolved cis-elements to circumvent the blockade to export of
unspliced transcripts from the nucleus. MPMV and other type D retroviruses, including simian
retrovirus-1 and 2 (SRV-1 and SRV-2), contain a small cis element, the constitutive transport element
(CTE), which is required for nuclear export of unspliced viral RNA [137,138]. When inserted into
unspliced or incompletely spliced HIV-1 transcripts, the MPMV CTE sequence replaces the function
Viruses 2013, 5 2773
of the Rev/RRE complex, leading to expression of Gag and Env followed by the production of
infectious virus particles [137]. Thus, Rev/RRE and the CTE provide similar roles in the nuclear
export of unspliced RNA in complex and simple retroviruses.
Insight into the mechanism by which CTE-containing RNAs are exported from the nucleus was
provided by proteomic studies that identified the host nuclear export protein Tip-associating
protein/Nuclear RNA export factor 1 (TAP/NXF1) as a binding partner of CTE complexes [139,140].
Microinjection of Xenopus oocyte nuclei expressing TAP/NXF1 and an intron containing the CTE
resulted in nuclear export of the RNA in the absence of splicing [141,142]. The TAP/NXF1 protein,
homologous to the mRNA export protein Mex67p in yeast, forms a heterodimer with NXT1 to
transport mRNAs out of the nucleus [139,140,143-145]. The N-terminal domain of TAP/NXF1 contains
an RNA recognition motif that binds to a structured stem-loop in the CTE, inducing structural changes in
both TAP/NXF1 and the CTE-containing RNA to promote nuclear export of the viral RNP [146].
Mutations in the RNA or in the coding region of TAP/NXF1 that disrupt CTE-TAP/NXF1 complex
formation prevent expression of CTE-containing reporters in vivo [146].
A putative cis-acting unspliced RNA transport element was also identified in RSV, which lies
within 115 nucleotide direct repeat (DR) sequences flanking the v-src oncogene [147]. DR elements
are highly conserved in avian retroviruses [148], and strains missing the src sequence maintain at least
a single DR element to remain replication-competent [149,150]. The biological role of the DR
elements is complex; pleotropic, contradictory effects on virus replication have been reported,
including differences in levels of cytoplasmic accumulation of RSV RNA, viral RNA stability,
expression of the Gag polyprotein, viral RNA packaging and virus assembly [148,151-153]. These
conflicting results may be explained by differences in cell types or the use of subviral reporter
constructs in some studies and full-length, replication-competent viruses in others.
RSV RNAs containing the DR elements are exported by the cellular mRNA export factor
TAP/NXF1 and the RNA helicase Dbp5 [139,154,155]. An additional host factor may bridge the
interaction because neither TAP/NXF1 nor Dbp5 bind the DR element directly. Because the RSV Gag
protein was reported to traffic through the nucleus [20], LeBlanc et al. used a subviral reporter
construct containing either the DR sequence or the sequence to examine whether Gag could enhance
translation by promoting nuclear export of unspliced RNA [155]. Gag did not enhance translation of
the reporter; however, nucleocytoplasmic fractionation of the RNA was not performed, so it is unclear
whether Gag had an effect on cytoplasmic levels of the DR- or -containing RNAs. Thus, these
experiments suggest that DR elements mediate nuclear export through TAP/NXF1 and Dbp5 to
stimulate translation of RSV unspliced RNA, but Gag is not likely to be involved in
DR-mediated RNA transport.
Taking the available data into account, we postulate that there is a temporal switch in RSV
replication, such that viral transcripts produced early after integration are exported using DR-mediated
interactions with Tap/NXF1 and Dbp5 to initiate the synthesis of Gag and GagPol proteins. We
hypothesize that as the levels of these viral structural proteins increase, the Gag protein enters the
nucleus where it may bind unspliced viral RNA and export it into the cytoplasm for encapsidation into
virions [20,156]. It is possible that other simple retroviruses may use a similar mechanism. We
speculate that MPMV RNA export could be similarly regulated, since a subpopulation of MPMV Gag
localizes to the nucleus and nuclear envelope, and the pp24 domain NLS has been linked to genomic
Viruses 2013, 5 2774
RNA incorporation [33-35]. Complex retroviruses regulate this temporal switch between early and late
gene expression differently; the Rev protein is synthesized from a fully spliced mRNA and then
traffics into the nucleus to promote nucleocytoplasmic transport of unspliced RNAs for structural gene
expression and genome packaging.
4. Foamy Viruses Use a Unique Pathway among Retroviruses for Nuclear Export of Viral RNAs
FVs, members of the genus Spumavirus, share similarities with both simple and complex
retroviruses, yet they have several unique characteristics (reviewed in [157,158]). Unlike other
retroviruses, Gag is the only protein translated from an unspliced transcript [159-161]. Additionally,
instead of being expressed as a fusion protein with Gag using frameshifting or termination codon
suppression, Pol is expressed from a separate spliced viral mRNA [162]. Similar to complex
retroviruses however, FVs encode the transcriptional transactivator protein Tas, which functions
analogously to HIV-1 Tat [163]. FVs do not encode an accessory protein with Rev-like functionality
[164], therefore, FVs rely entirely on host factors to mediate the export of unspliced RNA from the
nucleus, similar to the simple retroviruses MPMV and RSV.
Among retroviruses, PFV utilizes a unique set of cellular factors that bind to its viral RNA for
nucleocytoplasmic transport. Rather than using TAP/NXF1, like the cis-acting RSV and MPMV RNA
transport elements [139,155], spliced and unspliced PFV RNAs interact with host proteins HuR and
ANP32A/B to exit the nucleus through the CRM1 pathway [165]. HuR associates with PFV RNAs and
the adaptor proteins ANP32A or ANP32B, which bridge the association with CRM1 [165-167]. The
HuR-interacting RNA sequence likely resides in the 3’ region of the genome, which is shared among
spliced and unspliced PFV RNAs [165], although the PFV 3’ end does not share homology with
previously-characterized RNA sequences that bind HuR [165]. Although both the spliced and
unspliced PFV transcripts appear to be exported by the same pathway, subsequent interaction with the
cytoplasmic mRNA processing factor DDX6 may distinguish the genomic RNA from the viral
transcripts earmarked for translation on polysomes [168]. It is not yet known whether all PFV RNPs
exported by HuR-ANP32A/B-CRM1 traffic to the same subcellular location where DDX6 co-localizes
with genomic RNA in association with Gag. However, having spliced and unspliced RNAs targeted to
the same cytoplasmic localization may not pose a problem for selective genomic RNA selection by
PFV Gag because both segments of the bipartite cis packaging signal are present only on unspliced
viral RNA [169].
5. Nuclear Trafficking of Retroviral Gag Proteins: RSV as the Prototype
Historically, the Gag proteins of orthoretroviruses were thought to exist only in the cytoplasm of
infected cells. However, it has recently been shown that Gag proteins of diverse retroviruses localize to
the nucleus under specific conditions [19,20,24,30,72] (Figure 2). Demonstrating Gag nuclear
trafficking has been difficult because only a small portion of the total cellular Gag population is
detected in the nucleus under steady-state conditions. Despite this obstacle, the studies discussed
herein have yielded important insights into how the intranuclear localization of Gag proteins could be
involved in selection and encapsidation of the genomic RNA.
Viruses 2013, 5 2775
Figure 2. Localization of Gag proteins in and near the nucleus. The Gag proteins of
retroviruses and retrotranspons have been detected in the nucleoplasm (MLV, PFV and
RSV), in association with chromatin (PFV), in the nucleolus (FIV, HIV-1, MMTV, and
RSV), at the nuclear rim (MPMV, Ty1, FIV) and at pericentrosomal sites (HIV-1 and PFV).
The first retroviral Gag protein discovered to undergo active nucleocytoplasmic trafficking was
RSV Gag, which has a well-understood mechanism of nuclear entry and egress. Whereas under steady-
state conditions RSV Gag is detected primarily in the cytoplasm and along the plasma membrane,
treatment of RSV-infected or Gag-expressing cells with the CRM1-inhibitor leptomycin B (LMB)
dramatically concentrates Gag in the nucleus [20,170]. To gain insight into the role of RSV Gag in the
nucleus, a genetic approach was undertaken. Viruses encoding Gag mutants that bypass the nucleus
encapsidate reduced levels of genomic RNA and are noninfectious [156]. Reestablishment of Gag
nuclear trafficking by inserting a heterologous nuclear localization signal (NLS) into the MA domain
restores genome packaging to nearly wild-type levels [156]. In addition, in vitro evidence
demonstrated that binding of Gag to nucleic acids facilitates the association with the CRM1-RanGTP
export complex, suggesting that upon binding to nucleic acids in the nucleus, Gag is primed for export
[25]. Ongoing experiments to visualize the Gag-viral RNA interaction suggest that Gag co-localizes
with viral RNA in discrete subnuclear foci (Kaddis, Chiari-Fort and Parent, unpublished data).
Together, these results support a model in which RSV Gag selects the genomic RNA for encapsidation
in the nucleus (Figure 2).
To dissect the mechanism by which RSV Gag undergoes transient nuclear trafficking, a series of
experiments were performed to identify nuclear import and nuclear export signals (NES) in the RSV
Gag polyprotein [20,26,170]. Two independent NLSs were found in the RSV Gag protein, one in the
NC domain and the other in MA. The NLS in NC consists of a short stretch of basic residues, which is
typical of a classical monopartite NLS. This NLS binds directly to the adapter protein importin ,
which in turn recruits importin to import the Gag monomer through the nuclear pore complex [24-
26]. By contrast, the NLS in the RSV MA domain is atypical compared to other NLSs and resides
within the N-terminal 86 residues of Gag [26]. Instead of containing a discrete cluster of basic residues
like NC, there are 11 arginine and lysine residues dispersed throughout the sequence. However, in the
Viruses 2013, 5 2776
tertiary structure of the N-terminal MA sequence, these amino acids form a basic patch, presumably to
interact with the import factors transportin-3 (TNPO3) and importin-11 [26]. Interestingly, although
TNPO3 imports serine-arginine rich (SR) splicing factors into the nucleus, MA does not contain a SR-
rich region, indicating that the molecular basis underlying the TNPO3-Gag interaction is different from
other TNPO3 cargoes.
The MA domain does appear to be a critical determinant of RSV Gag function, as replacement of
the RSV MA sequence with HIV-1 MA abrogates nuclear trafficking of the RSV/HIV chimera [35].
The RSV/HIV chimeric mutant virus was able to replicate at a reduced level in a single-round
infectivity assay, suggesting that the HIV-1 MA domain may have a dominant effect, altering the
trafficking of RSV Gag and the mechanism of RNA packaging. Furthermore, it should be noted that
the RSV/HIV chimeric virus contained a reporter gene in place of the RSV env sequence, and the virus
was pseudotyped with the vesicular stomatitis virus G envelope protein, which may have affected the
infectivity results [35,171,172].
Why does RSV Gag contain two NLSs that interact with three different import factors? One
possibility is that Gag encodes redundant signals to ensure it enters the nucleus. Alternatively, each NLS
might be used selectively: the NLS that mediates entry of the Gag precursor during assembly could use
one or more sets of import factors whereas the MA and NC NLSs in the mature proteins could interact
with different importins during early infection. In support of this idea, insertion of a canonical NLS into
MA (in addition to the atypical NLS already present) interferes with infectivity [173]. The replication
defect does not involve budding, genome packaging, nuclear entry of the reverse transcription complex
or proviral DNA synthesis; instead, the mutant virus does not undergo integration [173]. Thus, it is
feasible that the nuclear import pathway followed by MA is crucial for successful integration, possibly
by properly targeting the PIC. It is intriguing to speculate that the same principle could apply to RSV
Gag during virus assembly: perhaps an importin bound to Gag directs it to a specific subnuclear
location. For example, TNPO3 binds to the MA region of Gag and is also required to transport SR-
protein splicing factors to splicing speckles [174-176]. If TNPO3 leads Gag to splicing speckles near
sites of ongoing mRNA transcription [115], Gag would be close to the viral RNA as it is synthesized
from the proviral DNA (Figure 1). We postulate that this strategy would be advantageous for Gag to
preferentially select unspliced viral RNA for packaging as it is being transcribed, particularly since
both spliced and unspliced RSV RNAs contain the cis-acting -packaging sequence. Experiments to
test this hypothesis are underway in our laboratory.
6. Localization of Retroviral Gag Proteins to the Nucleus and Nuclear Envelope
Although the mechanism of nuclear trafficking is the best understood for RSV Gag, other
orthoretroviral Gag proteins also localize to the nucleus or at the nuclear rim (Figures 2 and 3). The
MLV Gag protein is primarily localized in the cytoplasm and at the plasma membrane, although a
small nuclear pool (~18%) was detected using immunoelectron microscopy and biochemical
fractionation [38]. The authors proposed that the nuclear fraction of MLV Gag might play a role in
genome encapsidation or genomic RNA dimerization, and other studies support this hypothesis [177-
181]. Experiments performed in MLV-infected cells treated with the transcription inhibitor
actinomycin D demonstrated that unspliced viral RNA used for translation or packaging had different
Viruses 2013, 5 2777
half-lives [182-185], suggesting that MLV may use different nuclear export pathways to distinguish
translation-bound RNPs from encapsidated genomes. Interestingly, MLV genomic RNAs dimerize and
are co-packaged preferentially when the genomic RNA is transcribed from nearby chromosomal sites
[177-181], suggesting that MLV genomic RNA dimerizes co-transcriptionally within the nucleus.
Elegant experiments elucidated the structural basis for preferential binding of the dimeric MLV RNA
by showing that Gag binding sites on the RNA are exposed when dimerization occurs [186], thus
dimerization and packaging are linked for MLV. Finally, murine Y RNAs selectively encapsidated
into MLV particles are incorporated shortly after they are transcribed, raising the possibility that they
may be recruited in the nucleus [187]. Considered together, these studies raise the possibility that
selection of the dimeric genomic RNA and encapsidation of nuclear cellular RNAs are initiated by a
small nuclear population of MLV Gag, as suggested by others [38, 181, 187]. Alternatively, it is
feasible that one set of nuclear host factors binds to dimeric MLV genomes, transporting them to sites
of virion assembly in the cytoplasm, whereas a different complement of cellular RNA transporters may
direct monomeric unspliced RNA to ribosomes for translation. The merits of each model require
further investigation.
Figure 3. Subcellular sites of Gag-genomic RNA interaction where genomic RNA
packaging may be initiated prior to plasma membrane localization. Retroviral Gag-RNA
complexes have been visualized in pericentrosomal locations (HIV-1 and PFV) [168, 188,
189], on the cytoplasmic face of endosomes (HIV-1 and MLV) [190,191], on the
cytoplasmic face of the nuclear envelope (FIV, MPMV, and Ty1) [19,33,72,192], and
within the cytoplasm [193-196]. Genetic and biochemical data suggest that RSV Gag may
bind its genomic RNA in the nucleus during transient nuclear trafficking of the Gag
protein [20,26,156].
The lentiviral FIV Gag protein has a substantial degree of nuclear localization under steady-state
conditions in feline cells, its natural host [19] (Figure 2). Nearly all of the FIV Gag protein became
nuclear-localized when transfected or infected cells were treated with LMB, indicating that FIV Gag is
a nucleocytoplasmic shuttling protein, much like RSV Gag. Although the NLS and NES sequences in
FIV Gag have not been mapped, the LMB sensitive region of FIV Gag resides within the CA-NC-p2
Viruses 2013, 5 2778
sequence. When examined in HeLa cells, FIV genomic RNA and the Gag protein were observed at the
cytoplasmic face of the nuclear envelope in a Rev- and -dependent manner [72]. Deletion of the
sequence caused FIV RNA to be retained at the nuclear rim, whereas the Gag protein was localized to
the plasma membrane. These findings suggest that the site of interaction between FIV Gag and the
viral RNA may be the outer leaflet of the nuclear envelope (Figure 3). Kemler et al. raised the
possibility that FIV Gag may encapsidate its RNA genome in the nucleus, but because the efficiency
of export exceeds import, both Gag and the viral RNA genome appear to accumulate at the
cytoplasmic leaflet of the nuclear membrane [72].
In the same study, HIV-1 genomic RNA also accumulated at the nuclear envelope [72] (Figure 3).
However, the HIV-1 Gag protein was detected in the cytoplasm rather than at the nuclear rim,
suggesting that the Gag-genomic RNA interaction may be initiated in the cytoplasm. Other studies
reported that the pericentriolar microtubule organization center (MTOC) is a primary site of HIV-1
Gag-RNA interaction (Figure 3) [188,197]. The localization of HIV-1 RNA to the MTOC is mediated
by the host protein hnRNPA2, which is subsequently involved in transporting the HIV-1 RNP to the
plasma membrane for virion assembly [197,198]. The hnRNPA2 protein may initially bind to the
unspliced HIV-1 RNA in the nucleus, where it plays a role in pre-mRNA processing and alternative
splicing [199]. Thus, the MTOC may serve as a distribution center where HIV-1 Gag-RNA complexes
interact with the motor protein dynein before travelling along the microtubule network to the plasma
membrane [200]. In other studies of HIV-1 RNA trafficking in living cells, higher order Gag-RNA
complexes were detected primarily at the plasma membrane, although smaller oligomeric complexes
were found in the cytoplasm [194,196]. Therefore, although it remains unclear where HIV-1 Gag
initially binds to the genomic RNA, there is general agreement that viral RNP formation occurs prior
to plasma membrane localization (Figure 3).
Like FIV, a link may exist between MPMV Gag accumulation at the nuclear pore complex (NPC)
and genomic RNA packaging (Figures 2 and 3). The MPMV Gag protein interacts directly with the E2
SUMO conjugating enzyme Ubc9, which resides at the nuclear pore [34]. Additionally, overexpression
of Ubc9 results in co-localization of Gag with Ubc9 at the nuclear rim, suggesting that nuclear
trafficking of MPMV Gag may be a transient event mediated by interaction with Ubc9 [34].
MPMV Gag also accumulates in the nucleus with LMB treatment, indicating that it may undergo
CRM1-dependent nucleocytoplasmic shuttling [35]. MPMV Gag mutants with alterations in the NLS
in the pp24 domain no longer localize to the nuclear pore, and these mutant viruses are impaired in
genomic RNA packaging [33]. Together, these observations suggest that MPMV Gag trafficking to the
nuclear pore may be involved in viral RNA encapsidation.
The yeast long terminal repeat (LTR) retrotransposons Ty1 and Ty3 have some trafficking
properties in common with retroviruses. Ty1 and Ty3 encode Gag proteins that bind unspliced
genomic RNA to form virus-like particles. Mex67p, the yeast ortholog of TAP/NXF1, is required for
the export of Ty1 RNA from the nucleus [201]. In the cytoplasm, Ty1 RNA localizes with Gag to form
cytoplasmic foci called T-bodies [201,202]. Recently it was shown that efficient export of Ty1 RNA
from the nucleus is dependent on Gag [192]. Mutation of the gag initiation codon causes Ty1 RNA to
accumulate in the nucleus, where it is degraded, suggesting that Gag stabilizes the RNA. Expression of
Gag in trans restores Ty1 RNA nuclear export and RNA stability [192] (Figures 2 and 3). These data
suggest that Ty1 may use the Mex67p pathway for the bulk of Ty1 RNA nuclear export, whereas Gag
Viruses 2013, 5 2779
may transport the subset of the RNA used as the genome during virus-like particle assembly in
T-bodies. Ty1 Gag is not localized to the nucleus under steady-state conditions [201-204], but when
expressed in a mex67-temperature sensitive strain, Gag accumulates at the nuclear envelope in a subset
of cells [192]. Therefore, Gag nuclear localization appears to be transient, similarly to the nuclear
trafficking properties of RSV Gag [20,23-25,156]. In addition, a mutant of Ty3 that is defective in
RNA binding accumulates in the nucleus [205] and the Gag protein of Tf1, a retrotransposon of fission
yeast, is nuclear-localized under steady-state conditions [206] (Figure 2). Thus, retroviruses and
retrotransposons may share common mechanisms of Gag nuclear trafficking and unspliced RNA
export, suggesting that comparative studies of retroelements are likely to yield important insights into
novel mechanisms governing retrovirus particle assembly.
7. Localization of Gag to Subnuclear Sites
The Gag proteins of RSV and HIV-1 localize to the nucleolus under certain conditions [24] (Figure
2), and nucleolar trafficking of RSV Gag is dependent on basic residues in the NC domain. For RSV
NC NoLS activity resides in residues 3639 (KKRK), 6163 (RKR) and 70R/73K. By contrast, the
HIV-1 NC domain contains two independent NoLSs (R10/K11 and R32/K33/K34), each of which is
sufficient for nucleolar localization of NC [24]. HIV-1 NC localizes to the nucleus during
infection [57,58], but it is not clear whether nucleolar localization of the mature NC protein plays an
important role during early infection. It is intriguing that many capsid and nucleic acid binding
proteins from diverse viruses localize to the nucleolus, prompting the nucleolus to be referred to as
“the gateway to viral infection" [19,24,26,39,57,58,207].
Due to the transient nature of its trafficking through the nucleus, detecting RSV Gag in nucleoli
requires disruption of the nuclear export activity of Gag, either by mutating the p10 NES or treating
infected cells with LMB [24]. The retention of Gag in nucleoli is decreased in the presence of the RSV
Ψ packaging signal, suggesting that Gag binding to the packaging signal may either induce the Gag-
RNA complex to leave the nucleolus or may prevent trafficking of the RSV RNP through the
nucleolus. Whether there is a host protein or RNA that binds to RSV Gag-RNA complexes in the
nucleolus is unknown. A subset of the HIV-1 Gag protein also localizes to nucleoli when the provirus is
expressed at high levels from an inducible, Rev-dependent, integrated provirus. HIV-1 Gag accumulates
in nucleoli when co-expressed with Rev or NC, and positive FRET (fluorescence resonance energy
transfer) was observed between Gag and Rev, indicating an intimate association between these proteins
within the nucleolus [24]. Whether the nucleolar localization of HIV-1 Gag or the interaction between
Gag and Rev has any significant role in genomic RNA encapsidation or virus replication remains to be
Other retroviral Gag proteins have been reported to localize to the nucleolus under certain
conditions. For example, LMB treatment of transfected cells causes FIV Gag to accumulate in nucleoli
when expressed alone and nucleolar localization was dependent on NC [19] (Figure 2). However, the
importance of Gag or NC nucleolar localization was not further investigated. In addition, MMTV Gag
is partially nucleolar-localized in a murine mammary cell line chronically infected with a highly
tumorigenic strain of the virus [30]. Furthermore, a subset of MMTV Gag can be induced to
accumulate in the nucleolus with overexpression of ribosomal protein L9, which interacts with MMTV
Viruses 2013, 5 2780
Gag in an extraribosomal context (Figure 2). A functional role for this interaction was demonstrated by
knockdown of L9, which causes a decrease in MMTV particle production [30]. Thus, it is possible that
nucleolar trafficking of MMTV Gag is an important step in virus assembly pathway, although further
experiments will be needed to test this intriguing idea.
In addition to localizing to the nucleolus, the RSV Gag protein also accumulates in discrete
nucleoplasmic foci when restricted to the nucleus by mutating the NES or inhibiting CRM1-mediated
nuclear export [20,23] (Figure 2). It will be interesting to determine whether RSV Gag is associated
with a particular subnuclear body or tethered to a specific cellular protein or RNA at these sites. The
appearance of these subnuclear foci of RSV Gag is similar to the localization of Spumaretrovirus PFV
Gag in nucleoplasmic foci during prophase [82]. In the case of PFV, it was recently discovered that the
Gag protein binds to the host chromosome in mitotic cells using a chromatin-tethering domain encoded
in the glycine-arginine box II domain, which binds to core histones H2A and H2B [82]. The PFV Gag-
chromatin interaction facilitates proviral integration, although the precise mechanism has not been
defined. In addition, a CRM1-dependent nuclear export pathway was reported for PFV Gag, although
this finding is controversial [21,83]. An intriguing idea not yet experimentally tested is whether the
association PFV Gag with the proviral integration site [82] could position Gag at the site of PFV RNA
synthesis to facilitate selection of genomic RNA. Additional studies are required to determine whether
PFV Gag nuclear localization plays a role in genomic RNA encapsidation or whether its function is
restricted to early events, when it facilitates integration of the provirus.
8. Remaining Questions about Nuclear Trafficking during Retrovirus Assembly
In the past decade, great strides have been made in understanding the mechanisms that guide
nuclear import, nuclear export, and the intranuclear activities of retroviral and retrotransposon Gag
proteins. The use of a comparative approach to identify common and unique features of different Gag
proteins in the nucleus has been very illuminating. Although it is not yet clear whether all Gag proteins
transit through the nucleus at some low level, examination of the roles of nucleocytoplasmic
trafficking in those Gag proteins that do enter the nucleus or associate with the nuclear envelope (RSV,
MPMV, MLV, HIV-1, FIV, PFV, Ty1 and Tf1) are important to pursue.
Is Gag nuclear trafficking involved in genomic RNA packaging? To date, studies of RSV, MPMV,
and Ty1 support this idea. PFV Gag associates with chromatin and plays a role in integration, although
it might also function in genome encapsidation of nuclear transcripts [21,83]. In simple retroviruses,
there could be a temporal switch with unspliced viral RNA export mediated by Tap/NXF1 for
translation of structural proteins early after integration. As Gag proteins accumulate, they could enter
the nucleus and export viral RNA for packaging. Thus, could there be two pathways of nuclear export
of unspliced viral RNAs, or two distinct viral RNPs, one primed for packaging and the other for
translation? If so, it is logical to suggest that the cytoplasmic fates of retroviral RNAs may be pre-
determined in the nucleus according to the composition of the RNP.
Finding that several retroviral Gag proteins undergo nucleolar localization raises intriguing
questions about whether there is a unifying function of the nucleolus in virus replication. One
possibility is that retroviral Gag proteins interact with a host protein or RNA in the nucleolus involved
in genome packaging or virus assembly. It is intriguing that several RNA pol III transcripts are
Viruses 2013, 5 2781
enriched in retroviral particles, including RNAs that are processed in the nucleolus such as tRNA, U6
small nuclear RNA, 7SL [208,209], and 5S rRNA, [187,210-216]. Whether these RNAs are recruited
into virus particles as passengers encountered along the subcellular trafficking route of Gag or whether
they play a facilitating role in virus replication remains to be seen. As an interesting connection with
endogenous retroelements and the nucleolus, there is strong genetic evidence that the nucleolus may be
the site of RNP maturation for the LINE (long-interspersed nuclear element) retrotransposons, with U6
potentially playing a central role [217]. Thus, further investigation into the role of nuclear machinery
in retrovirus and retrotransposon RNA processing, nuclear export, and RNP trafficking may generate
new paradigms about genomic RNA encapsidation and virus particle assembly.
This work was supported by grants from the National Institutes of Health R01CA76534 (LJP),
P50GM103297 (LJP), F30 CA165774 (DVB), F31 CA171862 (RJK) and CURE Funding from the
Pennsylvania Department of Health (LJP). The Pennsylvania Department of Health specifically
disclaims responsibility for any analyses, interpretations, or conclusions of this project.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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... Historically, it was thought that recognition and binding of gRNA by Gag occurred in the cytoplasm or at the plasma membrane. However, given that retroviral RNA synthesis occurs in the nucleus and that the Gag proteins of many retroviruses, including Rous sarcoma virus (RSV) (1, 2, 10-15), feline immunodeficiency virus (16), foamy virus (17)(18)(19)(20)(21)(22)(23), human immunodeficiency virus type 1 (HIV-1) (13), Mason-Pfizer monkey virus (24)(25)(26), mouse mammary tumor virus (13,27), and murine leukemia virus (28), are present in the nucleus (29), it is plausible to hypothesize that retroviral Gag proteins associate with gRNA in the nucleus. ...
... The mechanisms governing Gag nuclear localization have been studied most extensively for RSV (1,2,(10)(11)(12)(13)(14)(29)(30)(31). Nuclear localization signals (NLSs) reside in the MA and NC domains, and a Crm1-dependent nuclear export signal (NES) was identified in the p10 domain upstream of CA (1, 10-12, 14, 31). ...
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Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.
... Gag oligomers that traffic toward the PM have been proposed in some studies [202]. A more provocative finding comes from the report of Gag trafficking to the nucleus, first reported for RSV Gag by the Parent laboratory [203,204]. For RSV Gag, nuclear localization has been linked to efficient packaging of gRNA, and it has been proposed that Gag is transiently imported into the nucleus where it collects gRNA, followed by nuclear export of the ribonucleoprotein complex and transit to the PM for assembly. ...
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The assembly of HIV-1 particles is a concerted and dynamic process that takes place on the plasma membrane of infected cells. An abundance of recent discoveries has advanced our understanding of the complex sequence of events leading to HIV-1 particle assembly, budding, and release. Structural studies have illuminated key features of assembly and maturation, including the dramatic structural transition that occurs between the immature Gag lattice and the formation of the mature viral capsid core. The critical role of inositol hexakisphosphate (IP6) in the assembly of both the immature and mature Gag lattice has been elucidated. The structural basis for selective packaging of genomic RNA into virions has been revealed. This review will provide an overview of the HIV-1 assembly process, with a focus on recent advances in the field, and will point out areas where questions remain that can benefit from future investigation.
... This protein accumulated in the cytoplasm at the plasma membrane, but also distributed to the nucleus. The Gag of multiple RVs is primarily found in the cytoplasm of infected cells, but for some RVs can be distributed to the nucleus, and in late stages of the viral life cycle accumulates at the plasma membrane for assembly [64]. ERV-L Gag has been found in the cytoplasm [65]. ...
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Background Retroviruses exist as exogenous infectious agents and as endogenous retroviruses (ERVs) integrated into host chromosomes. Such endogenous retroviruses (ERVs) are grouped into three classes roughly corresponding to the seven genera of infectious retroviruses: class I (gamma-, epsilonretroviruses), class II (alpha-, beta-, delta-, lentiretroviruses) and class III (spumaretroviruses). Some ERVs have counterparts among the known infectious retroviruses, while others represent paleovirological relics of extinct or undiscovered retroviruses. Results Here we identify an intact ERV in the Anuran amphibian, Xenopus tropicalis. XtERV-S has open reading frames (ORFs) for gag , pol (polymerase) and env (envelope) genes, with a small additional ORF in pol and a serine tRNA primer binding site. It has unusual features and domain relationships to known retroviruses. Analyses based on phylogeny and functional motifs establish that XtERV-S gag and pol genes are related to the ancient env -less class III ERV-L family but the surface subunit of env is unrelated to known retroviruses while its transmembrane subunit is class I-like. LTR constructs show transcriptional activity, and XtERV-S transcripts are detected in embryos after the maternal to zygotic mid-blastula transition and before the late tailbud stage. Tagged Gag protein shows typical subcellular localization. The presence of ORFs in all three protein-coding regions along with identical 5’ and 3’ LTRs (long terminal repeats) indicate this is a very recent germline acquisition. There are older, full-length, nonorthologous, defective copies in Xenopus laevis and the distantly related African bullfrog, Pyxicephalus adspersus. Additional older, internally deleted copies in X. tropicalis carry a 300 bp LTR substitution. Conclusions XtERV-S represents a genera-spanning member of the largely env -less class III ERV that has ancient and modern copies in Anurans . This provirus has an env ORF with a surface subunit unrelated to known retroviruses and a transmembrane subunit related to class I gammaretroviruses in sequence and organization, and is expressed in early embryogenesis. Additional XtERV-S-related but defective copies are present in X. tropicalis and other African frog taxa. XtERV-S is an unusual class III ERV variant, and it may represent an important transitional retroviral form that has been spreading in African frogs for tens of millions of years.
... Since αCentauri measurements were performed several hours after viral nuclear import, it was possible that PCA also occurred following nuclear entry of neosynthesized α-tagged IN. HIV-1 Gag and Gag-Pol polyproteins have been shown to traffic back to the nucleus and perinuclear area after translation in the cytoplasm (25). As a control, we therefore performed infections in the presence of an integrase inhibitor (raltegravir, RTG) to prevent de novo synthesis of viral proteins. ...
Significance The COVID-19 pandemic has highlighted the need to develop assays that can measure specific steps of viral replication on a large scale. Here we describe an innovative assay called αCentauri that uses fluorescence- or bioluminescence-based protein complementation assays to quantify the subcellular compartmentalization of viruses. As proof of concept, the Centauri fragment was tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment was attached to the integrase proteins of infectious HIV-1. Thereupon the trafficking of HIV to the nucleus efficiently reconstituted superfolder green fluorescent protein and NanoLuc αCentauri reporters. This technology offers a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening.
... Rex and Gag proteins participate in transport of proviral cDNA into cell nucleus. Note, Gag is also bound with centrosome proteins of spindle apparatus, which may lead to multicentric mitosis [33,34]. ...
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Diagnosis and prevention of the retroviral infection spread among farm animals still remain poorly developed primarily due to the fact that a hierarchic cascade of the events which underlie the retrovirus—host interactions involves molecular, intracellular levels, including cell organelles, and extracellular levels associated with the function of cellular immune networks. This paper presents an overview of own and literature data on the interaction of retroviral pathogen on the example of bovine leukemia virus (BLV) with intracellular structures of target cells. Here we consider four stages of the cascade of the events promoting pathogen, including i) introduction into the cell cytoplasm, ii) the synthesis of DNA copies of the viral genome RNA, iii) their transport into the cell nucleus, and iv) provirus DNA introduction into the host genome. The host genes interacting with viral structures are revealed at each stage. Two key processes contribute to genetic variability of retrovirus genome during infectious cycle: two viral RNAs dimerization needed for reverse transcription increases the frequency of recombination between RNA chains (N. Dubois et al., 2018), and provirus cDNA integration into the host genome can lead to activation of mutational and epigenetic events in both the pathogen genome and the host genome (A. Melamed et al., 2018). BLV pathogenesis is divided into two steps, the infectious cycle of mass infection of host target cells and sequential selection of individual infected cell clones. The peculiarity of the integration sites of the host genome is an increased frequency of mobile genetic elements originally closely related to exogenous retroviral infections (N.A. Gillet et al., 2013; T. Miyasaka et al., 2015). The high density of mobile genetic elements is characteristic of the host genomic DNA fragments flanked by inverted repeats of microsatellite AGC and identification sequence of the DNA transposon Helitron. The multiplicity of intracellular targets, whose polymorphism may be the basis of resistance to retroviral infections, allowed us to assume for the first time that the universal critical factor of the infectious process is the integration of proviral DNA into the host genome. It is suggested that the increased sensitivity of cells to productive BLV infection is due to a decrease in the activity of mechanisms involved in the genome protection from transposition activity. In the next communication, we will discuss the relationship between BLV-infected cells and host immune cell networks, which can also have a determining effect on the development of retroviral-induced infection. © 2018 Russian Academy of Agricultural Sciences. All Rights Reserved.
... Белки Rex и Gag участвуют в перемещении провирусной кДНК в ядро клетки. Важно, что Gag также связывается с белками центросом, организующими веретено деления, что может влиять на возникновения многополюсных митозов (33,34). ...
The retroviral Gag protein of human immunodeficiency virus type 1 (HIV-1) plays a central role in the selection of unspliced viral genomic RNA (gRNA) for packaging into new virions. Previously, we demonstrated that full-length HIV-1 Gag undergoes nuclear trafficking, where it associates with unspliced viral RNA (USvRNA) at transcription sites. To further examine the kinetics of HIV-1 Gag nuclear localization, we used biochemical and imaging techniques to determine the timing of HIV-1 entry into the nucleus. We also aimed to determine more precisely Gag’s subnuclear distribution to test the hypothesis that Gag associates with euchromatin, the transcriptionally active region of the nucleus. We observed that HIV-1 Gag localized to the nucleus at low expression levels shortly after its synthesis in the cytoplasm, suggesting that nuclear trafficking was not strictly concentration dependent. Furthermore, we found that HIV-1 Gag preferentially localized to the transcriptionally active euchromatin fraction compared to the heterochromatin-rich region in a latently infected T-cell line (J-Lat 10.6) treated with latency-reversal agents. Interestingly, HIV-1 Gag was more closely co-localized with euchromatin-associated histone marks near the nuclear periphery, the preferred location of HIV-1 proviral integration. Although the precise function of Gag’s association with histones in transcriptionally active chromatin regions remains uncertain, together with previous reports, this finding is consistent with a potential role for euchromatin-associated Gag molecules to initiate the selection of newly transcribed USvRNA in the nucleus for incorporation into virions. IMPORTANCE The traditional view of retrovirus assembly posits that packaging of gRNA by HIV-1 Gag occurs in the cytoplasm or at the plasma membrane. However, our previous studies showing that HIV-1 Gag enters the nucleus and binds to USvRNA at transcription sites suggest that gRNA selection may occur in the nucleus. In the present study, we observed that HIV-1 Gag trafficked to the nucleus and co-localized with USvRNA within 8 hours of expression. In infected T cells (J-Lat 10.6) reactivated from latency and in a HeLa cell line stably expressing an inducible Rev-dependent HIV-1 construct, we found that Gag preferentially localized with euchromatin histone marks associated with enhancer and promoter regions near the nuclear periphery, which is the favored site HIV-1 integration. These observations support the innovative hypothesis that HIV-1 Gag associates with euchromatin-associated histones to localize to active transcription sites, promoting capture of newly synthesized gRNA for packaging.
Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the virion intracellular assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.
During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. “Complex” retroviruses such as Human Immunodeficiency Virus (HIV) use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. “Simple” retroviruses such as Mason-Pfizer Monkey Virus (MPMV) and Murine Leukemia Virus (MLV) exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous Sarcoma Virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to WT virus and surprisingly, is replication competent albeit with a slower rate of spread compared to WT. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. Importance While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. RSV Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis- and trans-acting mechanisms for gRNA export.
RSV Gag nuclear entry is facilitated using three distinct host import factors that interact with nuclear localization signals in the Gag MA and NC domains. Here, we show that the MA region is required for nuclear import of Gag through the TNPO3 pathway. Gag nuclear entry does not require the CBD of TNPO3. Understanding the molecular basis for TNPO3-mediated nuclear trafficking of the RSV Gag protein may lead to a deeper appreciation for whether different import factors play distinct roles in retrovirus replication.
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Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus-like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1-less strain expressing galactose-inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense-mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2Δ mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci.
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The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy.
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HIV-1 Rev plays an important role in the late phase of HIV-1 replication, which facilitates export of unspliced viral mRNAs from the nucleus to cytoplasm in infected cells. Recent studies have shown that DDX1 and DDX3 are co-factors of Rev for the export of HIV-1 transcripts. In this report, we have demonstrated that DDX5 (p68), which is a multifunctional DEAD-box RNA helicase, functions as a new cellular co-factor of HIV-1 Rev. We found that DDX5 affects Rev function through the Rev-RRE axis and subsequently enhances HIV-1 replication. Confocal microscopy and co-immunoprecipitation analysis indicated that DDX5 binds to Rev and this interaction is largely dependent on RNA. If the DEAD-box motif of DDX5 is mutated, DDX5 loses almost all of its ability to bind to Rev, indicating that the DEAD-box motif of DDX5 is required for the interaction between DDX5 and Rev. Our data indicate that interference of DDX5-Rev interaction could reduce HIV-1 replication and potentially provide a new molecular target for anti-HIV-1 therapeutics.
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The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)1-23, human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA1-32, required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration.
Expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the presence of the viral trans-activator protein Rev. Rev is localized in the nucleus and binds specifically to the Rev response element (RRE) sequence in viral RNA. Furthermore, the interaction of the Rev activation domain with a cellular cofactor is essential for Rev function in vivo. Using cross-linking experiments and Biospecific Interaction Analysis (BIA) we identify eukaryotic initiation factor 5A (eIF-5A) as a cellular factor binding specifically to the HIV-1 Rev activation domain. Indirect immunofluorescence studies demonstrate that a significant fraction of eIF-5A localizes to the nucleus. We also provide evidence that Rev transactivation is functionally mediated by eIF-5A in Xenopus oocytes. Furthermore, we are able to block Rev function in mammalian cells by antisense inhibition of eIF-5A gene expression. Thus, regulation of HIV-1 gene expression by Rev involves the targeting of RRE-containing RNA to components of the cellular translation initiation complex.
Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions. Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein. This interaction is evolutionarily conserved as Yra1p also interacts with TAP. Conditional expression in yeast cells implicates Yra1p in the export of cellular mRNAs. Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants. The murine members of the family directly interact with TAP. Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs.
THE ancestors of the human immunodeficiency viruses (HIV-1 and HIV-2) may have evolved from a reservoir of African non-human primate lentiviruses, termed simian immunodeficiency viruses (SIV)1. None of the SIV strains characterized so far are closely related to HIV-12-6. HIV-2, however, is closely related to SIV (SIVmac) isolated from captive rhesus macaques (Macaca mulatta)7. SIV infection of feral Asian macaques has not been demonstrated by serological surveys8,9. Thus, macaques may have acquired SIV in captivity by cross-species transmission from an SIV-infected African primate. Sooty mangabeys (Cercocebm atys), an African primate species indigenous to West Africa, however, are infected with SIV (SIVsm) both in captivity9-11 and in the wild (P. Fultz, personal communication). We have molecularly cloned and sequenced SIVsm and report here that it is closely related to SIVmac and HIV-2. These results suggest that SIVsm has infected macaques in captivity and humans in West Africa and evolved as SIVmac and HIV-2, respectively.
RNA helicase plays an important role in host mRNA and viral mRNA transcription, transport, and translation. Many viruses utilize RNA helicases in their life cycle, while human immunodeficiency virus type 1 (HIV-1) does not encode an RNA helicase. Thus, host RNA helicase has been involved in HIV-1 replication. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether distinct DDX RNA helicases cross-talk and cooperate to modulate the HIV-1 Rev function. In this study, we noticed that distinct DDX RNA helicases, including DDX1, DDX3, DDX5, DDX17, DDX21, DDX56, except DDX6, bound to the Rev protein and they colocalized with Rev in nucleolus or nucleus. In this context, these DEAD-box RNA helicases except DDX6 markedly enhanced the HIV-1 Rev-dependent RNA export. Furthermore, DDX3 interacted with DDX5 and synergistically enhanced the Rev function. As well, combination of other distinct DDX RNA helicases cooperated to stimulate the Rev function. Altogether, these results suggest that distinct DDX DEAD-box RNA helicases cooperate to modulate the HIV-1 Rev function.