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1999 5: 1645-1655 RNA J. Yang and B. R. Cullen

constitutive transport element Structural and functional analysis of the avian leukemia virus

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© 1999 RNA Society

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Structural and functional analysis of the avian

leukemia virus constitutive transport element

JIN YANG and BRYAN R. CULLEN

Howard Hughes Medical Institute and Department of Genetics,Duke University Medical Center,

Durham,North Carolina 27710,USA

ABSTRACT

The observation that cells restrict the nuclear export of incompletely spliced transcripts via the canonical nuclear

mRNA export pathway implies that all retroviruses should have evolved a way to direct the unspliced form of their

genomic RNA into an alternate export pathway. While the Crm1-dependent pathway used by complex retroviruses to

export incompletely spliced viral transcripts is now fairly well understood, less is known about how simple retro-

viruses accomplish this task. However, the Mason±Pfizer monkey virus (MPMV) has been shown to encode a struc-

tured RNA sequence, termedtheconstitutive transport element (CTE),thatrecruits a cellular RNA export factor

termed Tap. Here we demonstrate that a CTE previously proposed to be present in the avian sarcoma/leukemia

(ASV/ALV) family of retroviruses indeed functions as a potent RNA export signal. We have mapped single- and

double-stranded regions present in the ASV/ALV CTE in vitro and report that this CTE is predicted to fold into a

structure bearing three distinct RNA stem-loops. However, only the central stem-loop is critical for CTE function and

this 69-nt structure is, in fact, sufficient when present as a dimer. While the ASV/ALV CTE is shown to function

independently of Crm1, as also previously reported for the MPMV CTE, it lacks any evident sequence homology to the

highly conserved MPMV CTE sequence. Together, these data define the secondary structure and biological activity of

an avian CTE sequence that may access a novel nuclear RNA export pathway. Keywords: CTE; nuclear export; retrovirus; RNA structure; RNA transport

INTRODUCTION

Retroviral replication requires the expression of un- spliced,fully spliced and,in some cases,partially spliced derivatives of the initial,genome-length transcript (re- viewed by Cullen,1998;Pollard & Malim,1998)+How- ever,eukaryotic cells have evolved mechanisms to prevent the nuclear export of RNAs that retain func- tional splice sites,that is,predominantly cellular pre- mRNAs+This presents retroviruses with a dilemma+On the one hand,retroviruses rely on the host cell to gen- erate spliced viral mRNAs and the initial,genome- length RNA must therefore contain recognizable splice sites+On the other hand,retroviruses also require the nuclear export of a portion of this same genome-length

RNA to express Gag and Pol and for packaging into

progeny virions+ It is now known that retroviruses have developed at least two distinct mechanisms to permit the nuclear

export of the unspliced and incompletely spliced RNAsthat are otherwise subject to nuclear retention by cel-lular splicing commitment factors (Cullen,1998;Pol-

lard & Malim,1998;Stutz & Rosbash,1998)+The best understood mechanism is the one utilized by human immunodeficiency virus type 1 (HIV-1) and several other complex retroviruses+HIV-1 encodes a regulatory pro- tein,termed Rev,that binds to,and multimerizes on,a structured RNA target site termed the Rev Response Element (RRE) found in incompletely spliced viral RNAs+ Rev contains a nuclear export signal (NES) that serves to recruit a cellular export factor termed Crm1 to the resultant ribonucleoprotein complex (Fornerod et al+,

1997a;Neville et al+,1997;Stade et al+,1997)+Crm1 in

turn targets these viral mRNAs to the nuclear pores and hence to the cytoplasm+

A second,distinct nuclear RNA export pathway is

utilized by Mason±Pfizer Monkey Virus (MPMV) (Bray et al+,1994)+MPMV is a simple retrovirus and therefore does not encode a regulatory protein comparable to

Rev+Nevertheless,MPMV faces the same dilemma of

how to move its genomic RNA from the nucleus to the cytoplasm+It is now apparent that MPMV also encodes a cis-acting structured RNA target site,in this case termed the constitutive transport element (CTE),that Reprint requests to:Bryan R+Cullen:Howard Hughes Medical Institute,Duke University Medical Center,Box 3025,Durham,North

Carolina 27710,USA;e-mail:culle002@mc+duke+edu+

RNA(1999),5:1645±1655+Cambridge UniversityPress+Printed in the USA+

Copyright  1999 RNA Society+

1645
on February 14, 2006 www.rnajournal.orgDownloaded from serves as the binding site for a host cell nuclear export factor termed Tap (Grîter et al+,1998;Braun et al+,

1999;Kang & Cullen,1999)+Tap appears able to target

CTE-containing viral RNAs to nuclear pores,and hence to the cytoplasm,and therefore serves a role some- what similar to Crm1 in HIV-1 RNA export (Katahira et al+,1999)+However,Crm1 and Tap clearly access distinct nuclear export pathways and appear to act via quite different mechanisms (Pasquinelli et al+,1997;

Saavedra et al+,1997;Kang & Cullen,1999)+

Although the use of retroviruses as a model system for the identification and study of novel nuclear RNA export pathways has therefore already facilitated the identification of the host cell nuclear export factors Tap and Crm1,it remains possible that retroviruses distinct from HIV-1 and MPMV may utilize yet other RNA ex- port pathways+In fact,with the exception of MPMV and the other simian type-D retroviruses,little is known about how simple retroviruses export their genomic RNA to the cytoplasm+However,in at least one other case (i+e+, the avian sarcoma/leukemia viruses),an RNAsequence that appears functionally analogous to the MPMV CTE has been identified (Ogert et al+,1996)+ Avian sarcoma viruses (ASV) differ from avian leu- kemia viruses (ALV) because of the presence of the src gene acquired by recombination with host sequences+ The srcgene is located between theenvgene and the

39LTR and,during recombination,the noncoding re-

gion present between envand the 39long terminal re- peat (LTR) in ALV has been duplicated+This sequence is therefore referred to as the direct repeat (DR),where

DR1 is located 59to

srcand DR2 39tosrc+Of course, in the ALV,only one copy of the DR exists (Fig+1)+

Previously,Ogert et al+(1996) showed that either

copy of the DR present in the Prague C (PrC) stain of ASV was able to partially substitute for the Rev:RRE axis in permitting the expression of the HIV-1 Gag pro- tein when present in cis+Deletion of both copies of the DR in ASV blocked viral replication,inhibited Gag pro- tein expression and,most importantly,reduced the cy- toplasmic expression of unspliced,but not of spliced,

ASV RNA+Although these observations strongly sug-

gest thatASV/ALV indeed contains a CTE,others haveargued that the ASV/ALV DR element is instead pri- marily required for the appropriate packaging of the retroviral genome into progeny virions and that effects on nuclear export of viral RNA are,at most,minor (Aschoff et al+,1999)+ In this manuscript,we report the structural and func- tional characterization of a DR sequence derived from the RAV-2 strain of ALV (Bizub et al+,1984) and dem- onstrate that this element is indeed able to mediate the nuclear export of an unspliced mRNA when present in cis+Using in vitro assays,we show that this CTE can fold into three RNA stem-loop structures and further show that it is the central stem that is most critical for ALV CTE function+Although the biological activity of the ALV CTE,like that of the MPMV CTE,was found to be independent of Crm1 function,our data also sug- gest that the mechanism of action of the ALV CTE may nevertheless be distinct from that of the MPMV CTE+

RESULTS

The ALV CTE induces nuclear RNA export

via a Crm1-independent pathway The CTE identified in DR2 of the PrC strain ofASV was previously localized to between nt 8770 and 8925 (Ogert et al+,1996)+To confirm that a functional CTE is also present in the ALV,which contain only a single DR,we subcloned the equivalent sequence from a previously described RAV-2 ALV isolate (Bizub et al+,1984) into the indicator construct pDM128/PL(Fridell et al+,1993)+

This 159-bp sequence extends 3 bp 39and 156 bp 59

to the 59boundary of theALV LTR and is 68% identical to the equivalent DR2 sequence in the PrC strain of ASV+

As previously described (Hope et al+,1990),the

pDM128 indicator plasmid contains a 59anda39splice site,derived from the HIV-1 envgene,flanking an in- tronic catindicator gene+pDM128/PL additionally con- tains a polylinker between the catgene and the 39 splice site,thus permitting the introduction of sequences of interest,such as the ALV CTE or,as previously de- scribed,the MPMV CTE or the HIV-1 RRE (Fridell et al+,

1993;Bogerd et al+,1998)+Because the

catgene is located between functional 59and 39splice sites,CAT enzyme expression is dependent on the nuclear export and translation of an unspliced mRNA,a process that, for the reasons described above,is normally very in- efficient+However,the introduction of an active CTE or of the HIV-1 RRE in cis(in the latter case in the pres- ence of Rev) results in the efficient nuclear export of this unspliced mRNAand,hence,in a marked induction in CAT expression+We therefore asked whether intro- duction of the putative ALV CTE would exert a similar effect+ Initially,we transfected the quail cell line QCl-3,which is permissive for RAV-2 replication (Cullen et al+,1983), FIGURE 1.Schematic representation of the genomic organization of ALV and ASV+The ASVs,such as Rous Sarcoma Virus,arose because of the recombinational acquisition of the host-cell-derived srcgene by ALVs+This resulted in a duplication of the 39noncoding sequence located between the envgene and the LTR in the ALVs and this duplicated sequence was hence referred to as the direct repeat+SD:splice donor;SA:splice acceptor+

1646J. Yang and B.R. Cullen

on February 14, 2006 www.rnajournal.orgDownloaded from with the parental pDM128/PL construct,with deriva- tives containing theALV CTE sequence in the sense or anti-sense orientation,or with a pDM128/PL derivative containing the MPMV CTE+As shown in Figure 2,the sense orientation ALV CTE potently activated CAT ex- pression from the pDM128 indicator plasmid,whereas the same ALV sequence in the anti-sense orientation had no detectable effect+As previously described (Kang & Cullen,1999),the MPMV CTE is not active in quail cells and the pDM128 construct containing this CTE was therefore also essentially inactive+

Whereas the MPMV CTE is active in human but not

quail cells,the PrC ASV CTE was initially reported to be active in avian cells but not primate cells (Ogert et al+,1996)+To examine the species tropism of theALV CTE,we compared the activity of these same four in- dicator plasmids upon transfection into the human cell

line 293T,the simian cell line COS,or the murine L cellline+As may be observed (Fig+2),both the ALV CTE

and the MPMV CTE proved to be equivalently active in all three of these distinct cell types+Therefore,based on this limited analysis,the ALV CTE does not display any obvious species tropism+In fact,recent unpub- lished experiments utilizing the PrC ASV CTE demon- strate that this CTE is also active in human cells when introduced into a pDM128-based indicator construct (R+Paca & K+Beemon,pers+comm+)+ To confirm that the ALV CTE is indeed acting at the level of unspliced nuclear RNA export,we transfected QCl-3 cells with pDM128 plasmids containing the ALV CTE inserted in either the sense or anti-sense orien- tation+At;48 h after transfection,the cells were har- vested and nuclear and cytoplasmic RNA fractions prepared+Analysis of these fractions using a previously described RNase protection assay (Kang & Cullen,

1999) revealed a dramatic increase in the level of un-

FIGURE 2.Species tropism of the ALV CTE+The indicator construct pDM128/ALV CTE was tested in cell lines from quail

(QCl-3),human (293T),monkey (COS-7),and mouse (L cell)+The parental pDM128/PL plasmid or a derivative with theALV

CTE in the antisense (AS) orientation served as negative controls+pDM128/MPMV CTE served as a positive control in

mammalian cells+COS-7 and QCl-3 cells were transfected using DEAE-Dextran and chloroquine with 25 ng of each

indicator construct,50 ng of pBC12/CMV/b-Gal,and 425 ng of pBC12/CMV per 35-mm culture+293T cells were transfected

using calcium phosphate with 50 ng of each indicator construct,25 ng of pBC12/CMV/b-Gal,and 925 ng of pBC12/CMV

per 35-mm culture+L cells were transfected using the FuGene 6 reagent with 200 ng of each indicator construct,800 ng of

pBC12/CMV/b-Gal,and 1mg of pBC12/CMV per 35-mm culture+Induced CAT activities were determined;48 h after

transfection and were adjusted for minor variations in transfection efficiency using theb-Gal internal control as previously

described (Bogerd et al+,1998)+

Analysis of ALV CTE function1647

on February 14, 2006 www.rnajournal.orgDownloaded from spliced cytoplasmic DM128 RNA in cells transfected with the sense orientationALV CTE plasmid,when com- pared to the antisense control (Fig+3,lanes 3 and 4)+In contrast,cytoplasmic spliced RNA levels were not sig- nificantly affected,whereas the ALV CTE had little if any effect on the pattern of nuclear RNA expression (Fig+3,lanes 1 and 2)+We therefore conclude that the

ALV CTE is indeed inducing pDM128-dependent CAT

expression by promoting the nuclear export of un- spliced catmRNA+

As noted above,the MPMV CTE directly recruits the

Tap nuclear export factor whereas the HIV-1 RRE:Rev complex instead recruits Crm1+Previously,we and oth- ers have shown that a truncated form of the Nup214/

CAN nucleoporin,termedDCAN,can directly bind to

Crm1 and block its ability to target the Rev:RRE com- plex to the nuclear pore (Fornerod et al+,1997b;Bo- gerd et al+,1998;Kang & Cullen,1999;Zolotukhin &

Felber,1999)+In contrast,DCAN exerts no effect on

Tap-dependent RNA export or on cellular mRNA ex-port+To examine whether Crm1 plays a role in medi- ating ALV CTE function,we tested the effect ofDCAN expression on ALV CTE or Rev:RRE function in trans- fected QCl-3 cells+As shown in Figure 4,DCAN effec- tively blocked Rev:RRE function,as expected,yet had no effect on the activity of the ALV CTE+Similar data were obtained using the drug leptomycin B,which is also a selective inhibitor of Crm1 function (data not shown)+We therefore conclude that the ALV CTE,like the MPMV CTE,functions independently of Crm1+

Structural analysis of the ALV CTE

As noted above,all known

cis-acting RNAexport signals share the property of being highly structured (Cullen,

1998;Pollard & Malim,1998)+To examine whether the

ALV CTE would also form one or more stable RNA

stem-loops,we transcribed the 159-ntALV CTE in vitro, end-labeled the CTE using T4 polynucleotide kinase, and then subjected the CTE to limited digestion using the double-strand specific V1 RNase or the single- strand specific RNases T1,T2,or A+Examples of the data obtained during this analysis are presented in Fig- ure 5A+In Figure 5B,we present a compilation of these data,obtained over several experiments,superimposed on a slightly modified,computer-predicted secondary structure of the CTE RNA sequence+

As may be observed,the ALV CTE is predicted to

fold into a secondary structure consisting of three dis- tinct RNA stem-loops,here designated SLI,SLII,and SLIII+The overall stability of this RNA structure is rea- sonable (DG5243+7 kcal/mol),although only the cen- tral SLII appears well folded+Thus,SLI displays only two strong double-stranded RNA hits and is only mod- estly stable (DG5213+8 kcal/mol),whereas SLIII,de- spite several strong double-stranded RNAhits,has only FIGURE 3.Analysis of RNA nuclear export in quail cells by ribonu- clease protection assay+QCl-3 cells were transfected with sense (S) or anti-sense (A) forms of pDM128/ALV CTE as described in Fig- ure 2+Cytoplasmic and nuclear RNA fractions were harvested from the cells at 48 h after transfection+A 208-nt 32

P-labeled probe (Kang

& Cullen,1999) containing a portion of the catgene,the 39splice acceptor site present in pDM128/PL,and an additional ªtagº se- quence was used in this assay to detect levels of the spliced (S) and unspliced (U) RNA+N-A:nuclear fraction from CTE antisense con- struct;C-A:cytoplasmic fraction from CTE antisense construct;N-S: nuclear fraction from CTE sense construct;C-S:cytoplasmic fraction from CTE sense construct+ FIGURE 4.Effect ofDCAN expression on ALV CTE function+Over- expression ofDCAN,the carboxy terminal region of nucleoporin NUP214/CAN,redirects the Crm1 nuclear export factor into the nu- cleoplasm and specifically inhibits Crm1-dependent nuclear export+ To examine if the ALV CTE utilizes this pathway,QCl-3 cells were transfected with 25 ng of pDM128/ALV CTE in the presence of 425 ng of pBC12/CMV/DCAN,or of pBC12/CMV as a control+Similarly,QCl-3 cells were also transfected with pDM128/RRE (25 ng) in the pres- ence or absence of pcRev (25 ng) and of 400 ng of pBC12/CMV/

DCAN or pBC12/CMV+

1648J. Yang and B.R. Cullen

on February 14, 2006 www.rnajournal.orgDownloaded from

FIGURE 5.Secondary structure of the ALV CTE+A:The full-length ALV CTE was in vitro transcribed,59end-labeled and

finally subjected to limited RNase digestion+AH:alkaline hydrolysis for generation of a continuous RNAladder;U2:complete

RNase U2 digestion for diagnostic A residues along the CTE;V1:RNase V1,a double-stranded RNase with no apparent

sequence specificity;T1:a single-stranded RNase that cleaves on the 39side of G residues;T2:cleaves RNAon the 39side

of A residues in single-stranded regions;A:RNase A cuts RNA at single-stranded regions at C or U residues under high salt

conditions+First Load gives clear resolution of the 39half of the RNA molecule whereas the Second Load allows analysis

of the 59half of the RNA+Diagnostic residues are labeled at the side of the autoradiograph with the same arbitrary

numbering used inB+B:The predicted secondary structure of the ALV CTE+The structure shown here is based on both

RNase digestion data and on a computer-predicted secondary structure model derived from the RNA folding server ªmfold

serverº (Dr+Michael Zuker,Institute for Biomedical Computing,Washington University;http://www+ibc+wustl+edu/;zuker/)+

This predicted structure was then slightly modified to accommodate the RNase digestion data+The open and closed arrows

indicate specific cleavages induced by the single-strand- or double-strand-specific RNases tested inA+

Analysis of ALV CTE function1649

on February 14, 2006 www.rnajournal.orgDownloaded from a modest predicted stability (DG525+7 kcal/mol)+In contrast,SLII displays a striking 10-bp perfect pre- dicted RNA duplex at its base that appears,not sur- prisingly,to be effectively formed in vitro (Fig+5A)+ Although the apical region of SLII displays significantly less structure,with only one strong and several weak double-stranded RNA hits,this RNA stem-loop is nev- ertheless predicted to be quite stable (DG5228+2 kcal/mol)+The overall validity of this structure is sup- ported not only by the detection of double-strand spe- cific cleavages in several areas of predicted helical structure,but also by the selective detection of strong single-strand specific cleavages in predicted bulge or loop structures in all three stem-loops (Fig+5)+

The ALV stem-loop II sequence

is critical for CTE function To test whether the ALV CTE secondary structure pro- posed in Figure 5B has any physiological significance, we first performed deletions to test the relevance of each of the three proposed stem-loops for ALV CTE function+As shown in Figure 6,deletion of all of SLI or all of SLIII reduced,but did not eliminate,ALV CTE function in QCl-3 cells+We therefore next asked if SLII alone would be sufficient for biological activity+In fact, however,a single copy of the 69-nt SLII sequence proved essentially inactive (Fig+6)+ Previously,several groups have reported that the pres- ence of a single nuclear RNA export factor binding site is not sufficient for biological activity in vivo+Thus,a single copy of the RRE stem-loop II binding site for

Rev,of the stem-loop D binding site for the human

T-cell leukemia virus (HTLV) Rex protein,or of the min-

imal Tap binding site on the MPMV CTE is not sufficientto induce the detectable nuclear export of an unsplicedRNA,while two,duplicated copies promote export ef-

ficiently (Huang et al+,1991;Graene et al+,1994;Kang &

Cullen,1999)+We therefore wondered if two tandem

copies of ALV CTE SLII would give a similar result+In fact,as shown in Figure 7,two tandem copies of SLII were as active as the full-length ALV CTE in inducing pDM128-dependent CAT expression in both QCl-3 and

293T cells+Perhaps surprisingly,introduction of a third

copy of SLII had no additional beneficial effect+ To confirm the critical importance of the SLII se- quence for ALV CTE function,we next performed a limited mutational analysis of SLII in the context of the full-length ALV CTE sequence+Initially,we focused on the perfect 10-bp inverted repeat that forms the pre- dicted base of the SLII structure+Mutations M1 and M2 (Fig+8) were designed to disrupt the first 5 bp of this predicted helix but were also designed to restore the full-length helix when introduced simultaneously+As shown in Figure 9A,both M1 and M2 inhibited ALV CTE function by approximately twofold,whereas intro- duction of both mutations simultaneously (M112) fully restored wild-type activity+ FIGURE 6.Comparison of the full length ALV CTE with truncated derivatives lacking SLI,SLIII,or both SLI and SLIII,as defined in Figure 5B+The corresponding ALV CTE sequences were cloned into the pDM128/PL indicator plasmid in the same way as the full-length CTE+The resultant indicator constructs were transfected into QCl-3 cells and the induced CAT activity determined as described in Fig- ure 2+ FIGURE 7.The 69-nt SLII sequence represents the core ALV CTE target structure+Up to three copies of the SLII sequence were se- quentially cloned into the pDM128/PLpolylinker region,as describedquotesdbs_dbs13.pdfusesText_19

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