HIVmay produce inhibitory microRNAs (miRNAs) that block
?Corresponding author.Tel.:+15173556475x1101; fax:+15173555125.
E-mail addresses:coutur14@https://www.360docs.net/doc/8c3025610.html,(J.P.Couturier), rootbern@https://www.360docs.net/doc/8c3025610.html,(R.S.Root-Bernstein).
counterpart receptors,B7-1(CD80)and B7-2(CD86)on antigen-presenting cells(APC).Noteworthy events asso-ciated with CD28binding to its ligands(Fig.1)include T-cell proliferation and differentiation into memory and effector cells,secretion of cytokines such as IL-2,IFN-g; GM-CSF and TNF-a;and prevention of anergy and apoptosis(Bour-Jordan and Bluestone,2002;Allison, 1994;Linsley and Ledbetter,1993).One would expect that interference with CD28expression would therefore lead to decreased cytokine production,anergy and apoptosis,all of which would signi?cantly impair T-cell regulated functions,including activation of APCs and B cells. Indeed,during HIV-1infection,the expression of CD28signi?cantly decreases(Gamberg et al.,2004; Park et al.,1998;Kammerer et al.,1996;Haffar et al., 1995;Caruso et al.,1994),subsequently resulting in T-cell dysregulation such as abnormal cytokine produc-tion,anergy and apoptosis(Linsley and Ledbetter,1993; Groux et al.,1992).The precise mechanism of CD28 down-regulation during HIV-1infection has yet to be fully elucidated,but the discovery of viral-encoded miRNA from Epstein-Barr virus(EBV)(Pfeffer et al., 2004)offers a new venue of possibilities.
Since the discovery of their existence,numerous miRNAs have been elucidated from many eukaryotes ranging from nematodes to humans(Ambros et al., 2003;Lagos-Quintana et al.,2003;Lewis et al.,2003; Lim et al.,2003;Lagos-Quintana et al.,2001;Lee and Ambros,2001).It was shown that partial complemen-tarity between miRNAs and their mRNA target(s)was suf?cient to suppress protein translation(Doench et al., 2003;Saxena et al.,2003;Zeng et al.,2002;Hutvagner and Zamore,2002;Ambros,2001;Lagos-Quintana et al.,2001),and very recently it was veri?ed that viruses could also encode miRNAs(Pfeffer et al.,2004).This discovery inevitably provokes the question of how many other viruses could encode miRNAs and to what extent viral miRNA affects host cell protein expression.It also raises the prospect of a correlation between genomic complementarity and a higher order process such as microRNA:mRNA interactions.
Genomic complementarity between virus and host might confer a unique advantage to HIV-1and other intracellular pathogens as well.In light of these recent discoveries,we hypothesize that HIV-1may produce miRNA that directly alters CD4+T-cell,macrophage
(B)
Fig.1.Key activities associated to T-cell-APC binding(T-cell activation)with(A)or in the absence of(B)CD28co-stimulation.Normal immune function is clearly dependent upon the presence of CD28on CD4+T cells.Interaction of CD28with its counterpart B7-1/B7-2receptors on APCs provides the CD4+T cell with a co-stimulatory signal required for T-cell proliferation and differentiation.The consequences of CD28-negative T cells also affect the proper functioning of other immune system cells.Of particular importance to the current paper is the fact that interference with CD28expression can raise CTLA-4expression.
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and dendritic cell activity.To initiate an assessment as to the validity of this hypothesis,alignments between the HIV-1proviral genome and the mRNA cds of various proteins and interleukins produced by CD4+T cells and macrophages(CD28,CTLA-4,CD4,CD40L, TNF-a;TNF-b;TGF-b1;IL-1b;IL-2,IL-3,IL-4,IL-6,IL-10,IL-12,IL-13,IL-15,IL-16and IL-18)were conducted to identify regions of complementarity. Although much has been documented regarding miRNA and target mRNA interactions,the speci?c requirements of miRNA:target mRNA binding and determination of the subsequent effects(mRNA degra-dation or translational inhibition)require further investigation.Many reports have shown that transla-tional repression occurs when the miRNA binds to the 30UTR of its mRNA target(Lee et al.,1993).Other reports indicate that binding and subsequent transla-tional repression is not restricted to the30UTR and can occur within the cds of a gene(Kawasaki and Taira, 2003;Saxena et al.,2003).In other words,the target sequence could be virtually anywhere within the mRNA (Pasquinelli,2002).Also,if many separate,partially complementary regions of binding exist between the miRNA and the target mRNA,the miRNA could exert its effects in a synergistic manner that involves the individual regions of partial complementarity(Novina et al.,2002;Doench et al.,2003;Zeng et al.,2002,2003; Hutvagner and Zamore,2002).Furthermore,based on certain statistical parameters,a$21-nucleotide(nt) RNA may partially bind to mRNAs containing$6–8 consecutive base pairs(Novina et al.,2002)and it has also been demonstrated that siRNA can directly inhibit the expression of off-target mRNAs with sequence similarity involving as low as10contiguous nucleotides (Jackson et al.,2003).Based on these?ndings,we did not expect the regions of complementarity to be restricted to a few locations between the HIV-1proviral genome and the mRNA cds,but rather to be present in multiple areas as suggested in Tables1and2,and Figs. 2and3.
In this report,we present regions of complementarity (with remarkable correlation to the previously men-tioned research?ndings regarding miRNA:target mRNA interactions)between the proviral genome of HIV-1and the mRNA cds of proteins and interleukins produced by CD4+T cells and macrophages.The clinical signi?cance of these results remain the subject of further investigation.
2.Methods
A nucleic acid sequence local alignment tool(http:// pbil.univ-lyon1.fr/lfasta.php)employing a modi?ed Needleman–Wunsch and Smith–Waterman algorithm (Pearson and Lipman,1988)was used to identify regions of complementarity.The LFasta parameters were set at default settings:‘‘Number of alignments’’(20),‘‘Penalty for the?rst residue in a gap’’(à12)and‘‘Penalty for additional residues in a gap’’(à2).For the‘‘Strand’’option,both sequences were aligned as[Direct].The HIV-1proviral genome was inputted as‘‘Sequence1’’and the complementary sequence of each protein/ interleukin mRNA cds was inputted as‘‘Sequence2’’. The HIV-1proviral genome was acquired from the EMBL-Bank database(https://www.360docs.net/doc/8c3025610.html,/genomes/ virus.html):Accession number(AC)X01762(Muesing et al.,1985;Wong-Staal et al.,1985).The mRNA cds of each CD4+T-cell and macrophage protein/interleukin was acquired from the GenBank database:CD28-AC J02988(Aruffo and Seed,1987),CD4-AC M12807 (Maddon et al.,1985),CTLA-4-AC AF414120(Wu and Ling,2001),CD40L-AC L07414(Gauchat et al.,1993), IL-1b-AC M15330(Nishida et al.,1987),IL-2-AC S77834(Eizenberg et al.,1995),IL-3-AC M14743(Yang et al.,1986),IL-4-AC M13982(Yokota et al.,1986),IL-6-AC M14584(May et al.,1986),IL-10-AC M57627 (Vieira et al.,1991),IL-12(a chain)-AC M65271 (Gubler et al.,1991),IL-12(b chain)-AC M65272 (Gubler et al.,1991),IL-13-AC L06801(Mckenzie et al.,1993),IL-15-AC U14407(Grabstein et al.,1994), IL-16-AC AF053412(Du et al.,1999),IL-18-AC AY044641(Liu et al.,2001),TGF-b1-AC BC022242 (Strausberg et al.,2002),TNF-a-AC BC028148(Straus-berg et al.,2002)and TNF-b-AC D00102(Kobayashi et al.,1986).The complementary sequences of the protein/interleukin mRNA cds were acquired utilizing the complement acquisition feature at the Sequence Manipulation Suite website(http://www.ualberta.ca/ $stothard/javascript/index.html).Thus,RNA comple-mentarity is expressed in our study in terms of homologies between the proviral HIV-1genome and the complementary sequences of host protein cDNA. The complementary sequence of each CD4+T-cell and macrophage protein/interleukin mRNA cds(entire cds sequence)was aligned directly with1000-nt segments of the HIV-1proviral genome,and the reported identities(i.e.homologies)were considered regions of complementarity between these2nt sequences.The HIV-1proviral genome was segmented into1000-nt alignment windows by two methods:(A)1–1000,1001–2000,2001–3000,3001–4000,4001–5000,5001–6000, 6001–7000,7001–8000,8001–9000,9001–9748,and(B) 1–500,501–1500,1501–2500,2501–3500,3501–4500, 4501–5500,5501–6500,6501–7500,7501–8500,8501–9500,9501–9748.The reading frame of the genes was ignored,since the mechanism of RNAi action does not require the complementarity to be reading-frame appro-priate(Kawasaki and Taira,2003;Saxena et al.,2003). Blockage of RNA transcription simply requires segmen-ted binding of an RNA to its complement.The results were represented in LFasta format(Pearson and
J.P.Couturier,R.S.Root-Bernstein/Journal of Theoretical Biology235(2005)169–184171
LFasta output from alignment between HIV-1 (Seq1)proviral genome nt 7001-8000 and complementary sequence of CD28(Seq2)1514nt mRNA cds.
46.8% identity in 391 nt overlap; init: 45, opt: 51 40 50 60 70 80 90
Seq1 GACAATTTACCGTCAGACCGTCTTCTTCTCCATCATTAATCTAGACGGTTAAAGTGTCTG :::: :::: :::: :: :::::: : : X:::::: : Seq2 GACATTTTAGTTGCAGAAGAAAGGCTAGGAAATCATTCCTTT---TGGTTAAATGGGTGT 1130 1140 1150 1160 1170 1180 100 110 120 130 140 150 Seq1 TTACGATTTTGGTATTATCATGTCGACTTGGTTAGACATCTTTAATTAACATGTTCTGGG ::: ::::::X : : : :: ::: :: : : : : : Seq2 TTAATCTTTTGGT-TAGTGGGTTAAACGGGGTAAGTTAGAGTAGGGGGAGGGATAGGAAG 1190 1200 1210 1220 1230 1240 160 170 180 190 200 Seq1 TTGTTGTTATGTTCTTTTTCATAGGCATAGGTCTCTCCTG--GTCCCTCTCGTA--AACA : ::: : :: : : : ::: :: : : :::: : Seq2 ACATATTTAAAAACCATTAAAACACTGTCTCCCACTCATGAAATGAGCCACGTAGTTCCT 1250 1260 1270 1280 1290 1300 210 220 230 240 250 260 Seq1 ATGTTATCCTTTTTATCCTTTATACTCTGTTCGTGTA-ACATTGT-AATCATCTCGTTTT :: : :: :: ::: ::::::: : :: ::::::: : :: : : Seq2 ATTTAATGCTGTTT-TCCTTTAGTTTAGAAATACATAGACATTGTCTTTTATGAATTCTG 1310 1320 1330 1340 1350 1360 270 280 290 300 310 320 Seq1 ACCTTATTGTGAAATTTTGTCTATCT-ATCGTTTAATTCTCTTGTTAAACCTTTATTATT : : :::: : :::::: : : : : : :: :: :: : : : : Seq2 ATCATATTTAGTCATTTTGACCAAATGAGGGATTTGGTCAAATGAGGGA-TTCCCTCAAA 1370 1380 1390 1400 1410 330 340 350 360 370 380 Seq1 TTGTTATTAG--AAATTCGTCAGGAGTCCTC-CCCTGGGTCTTTAACATTGCGTGTCAAA ::: :: ::: : ::::: : : ::: : : :: :::: :: Seq2 GCAATATCAGGTAAACCAAGTTGCTTTCCTCACTCCCTGTCATGAGACTTCAGTGTTAAT 1420 1430 1440 1450 1460 1470 390 400 410 Seq1 ATTAACACCTCCCCTTAAAAAGATGACATTA :: ::: : :: ::::: : : :: Seq2 GTTCACAATATACTTTCGAAAGAATAAAATA 1480 1490 1500 1510
LFasta output from alignment between HIV-1 (Seq1)proviral genome nt 9001-9748 and complementary sequence of CD28(Seq2)1514nt mRNA cds.
? *
* ? 44.9% identity in 379 nt overlap; init: 40, opt: 46
10 20 30 40 50 60
Seq1 TCTCCTCCTCCTCCACCCAAAAGGTCAGTGTGGAGTCCATGGAAATTCTGGTTACTGAAT : X::::::::X ::: : : :: :: : :: :: : :: : : :: : Seq2 TATCCTCCTCCT-TACCTA----GACAATGAGAAGAGCAATGGAACCATTATCCATG--T 460 470 480 490 500 70 80 90 100 110 120 Seq1 GTTCCGTCGACATC-TAGAATCGGTGAAAAATTTTCTTTTCC-CCCCTGACCTTCCCGAT : : ::: : : : :: :::: : :: : : :::: : : Seq2 GAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGT 510 520 530 540 550 560 130 140 150 160 170 180 Seq1 -TAAGTGAGGGTTGCTTCTGTTCT-ATAGGAACTAGACACCTAG--ATGGTGTGTGTTCC ::: ::::: : :: :: : ::: :::: : ::: :: Seq2 GCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTAT 570 580 590 600 610 620 190 200 210 220 230 Seq1 GATGAAGGGACTAATCGTCTTGATGTG-TGGTCCCGGTCCCTAGTCTATAGG----TGAC :: : : : :: : : :: :: : : : ::: : : :::: Seq2 TATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGAC 630 640 650 660 670 680 240 250 260 270 280 290 Seq1 TGGAAACCTACCACGATGTTCGATCATGGTCAACTCGGTCTCTTCAATCTTCTTCGGTTG : :: :: : : :: : :: : :: : : : : :: Seq2 TCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGC--CCCACCACGCGAC 690 700 710 720 730 740 300 310 320 330 340 350 Seq1 TTTCCTCTCTTGTGGTCGAACAATGTGGGACACTCGGACGTACCTTACCTACTGG--GCC :: : :: : :: : : : : :: : : : :: :::: ::: Seq2 TTCGCAGCCTATCGCTCCTGACACGGACGCCTATC--CAGAAGCCAGCCGGCTGGCAGCC 750 760 770 780 790 360 370 Seq1 TCTCTCTTCACAATCTCAC : ::: : :::: :::: Seq2 CCCATCTGCTCAATATCAC 800 810
*Initial region: Score based on algorithm used to identify the best initial regions of identity.
?Optimized score: Score based on optimally joined initial regions of identity.
(Pearson et al ., 1988).
Fig.2.Two LFasta alignment results between the HIV-1proviral genome and the complementary sequence of the mRNA cds of CD28.In boxes are regions of consecutive base-pairing of nucleotides representing potential miRNA:mRNA interaction based on criteria from literature reports.The HIV-1nucleotide alignment window for each result is indicated.
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Lipman,1988).Fig.2depicts representative LFasta results between HIV-1and CD28,and Fig.3depicts LFasta results between HIV-1and IL-2,CD40L and CD4.
When performing sequence comparisons between proteins or nucleic acids,frequent matters of debate often involve choices of the particular alignment tool used,the sequence lengths compared and the parameter settings.Short alignments of less than 18nt or less than 40%identity were discarded from our results since studies of miRNAs show that they must (in general)be 418–20nt with no more than 3–4mismatches to result in translational inhibition (Saxena et al.,2003;Zeng et al.,2002,2003;Ambros,2001;Lee et al.,1993).However,more recent reports suggest that this is not a strict criterion for miRNA activity (Novina et al.,2002;Doench et al.,2003;Jackson et al.,2003;Zeng et al.,2002,2003;Hutvagner and Zamore,2002).Our objective was to identify relatively small (o 30nt)regions of high complementarity with strong correlation
LFasta output from alignment between HIV-1 (Seq1)proviral genome nt 3001-4000 and complementary sequence of IL-2 (Seq2) 812 nt mRNA cds.
80.0% identity in 20 nt overlap; init: 45, opt: 52 420 430 Seq1 TTAATACATTTGAGGAATCT X::::: :::::X ::: : Seq2 TTAATAAATTTGATAAATAT 770 780
LFasta output from alignment between HIV-1 (Seq1)proviral genome nt 4001-5000 and complementary sequence of IL-2 (Seq2) 812 nt mRNA cds.
90.0% identity in 20 nt overlap; init: 66, opt: 66 920 930 Seq1 TTTAAGTTTTAAAAGCCCAA X:::::::::: : :::::X Seq2 TTTAAGTTTTACATGCCCAA 240 250
LFasta output from alignment between HIV-1 (Seq1)proviral genome nt 4001-5000 and complementary sequence of CD40L (Seq2) 1816nt mRNA cds.
83.3% identity in 24 nt overlap; init: 67, opt: 68 150 160 Seq1 TTATTATCTCGTCAATTATTTTTT : X:::::: :: : ::::::::X Seq2 TAATTATCTTGTTATTTATTTTTT 1770 1780
LFasta output from alignment between HIV-1 (Seq1) proviral genome nt 2001-3000 and complementary sequence of CD4 (Seq2) 1742 nt mRNA cds.
81.0% identity in 21 nt overlap; init: 44, opt: 56 700 710 Seq1 CATGTCTTTACCTTTTCCTTC :: : : :: X:::::::::X Seq2 CACGCCATTTCCTTTTCCTTC 1670 1680
LFasta output from alignment between HIV-1 (Seq1)proviral genome nt 8001-9000 and complementary sequence of CD4 (Seq2) 1742 nt mRNA cds.
63.0% identity in 81 nt overlap; init: 44, opt: 87
80 90 100 110 120 130 Seq1
GAACCTTACGATCAACCTCATTAT-TTAGAGACCTTGTCTAAACCTTATTGTACTGGACC ::: :: :: :: :::::: : :: ::::: :: : : : : :::::
Seq2 GAAGCTCCCGCTCCACCTCACCCTGCCCCAGGCCTTGCCTCAGTATGCTGGCTCTGGA-- 910 920 930 940 950 960 140 150 Seq1 TACCTCACCCTGTCTCTTTAA X:::::::::X : ::: :: Seq2 AACCTCACCCTGGCCCTTGAA 970 980
*Initial region: Score based on algorithm used to identify the best initial regions of identity.
?Optimized score: Score based on optimally joined initial regions of identity.
(Pearson et al ., 1988).
(A)
(B)
(C)
(D)
(E)
Fig.3.LFasta alignment results between the HIV-1proviral genome and the complementary sequence of the mRNA cds of IL-2(A,B),CD40L (C)and CD4(D,E).In boxes are regions of consecutive base-pairing of nucleotides representing potential miRNA:mRNA interaction based on criteria from literature reports.The HIV-1nucleotide alignment window for each result is indicated.
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to literature reports of siRNA:target mRNA interac-tion,so the method of aligning1000-nt segments of the HIV-1proviral genome against the mRNA cds of each protein and interleukin was deemed appropriate for the particular alignment algorithm utilized and to ensure adequate scanning of the HIV-1genome for regions of complementarity.
Based on research?ndings of miRNA and siRNA activity(Novina et al.,2002;Doench et al.,2003; Jackson et al.,2003;Saxena et al.,2003;Zeng et al., 2002,2003;Hutvagner and Zamore,2002;Lagos-Quintana et al.,2001;Lee et al.,1993),we established our criteria for signi?cant alignments as follows: 475.0%in20–30nt overlap,470.0%in31–40nt overlap,465.0%in41–60nt overlap,460.0%in 61–80nt overlap,455.0%in81–100nt overlap and 440.0%in4101nt overlap.Application of these stringent criteria excluded many shorter alignments and alignments with low complementarity,and was considered to be an appropriate baseline from which to identify potentially signi?cant alignments.
We also validated our criteria by using a web-based program from Dharmacon Inc.(‘‘siDESIGN Center’’at https://www.360docs.net/doc/8c3025610.html,/rnadesign/default.aspx) to identify multiple siRNA target regions within each protein mRNA examined in this report.Most of these target regions corresponded exactly to sequences within the regions of complementarity reported in Tables1–3, suggesting that the results reported below are not highly dependent on any particular alignment strategy or choice of signi?cance criteria.
3.Results
Tables1–3list the alignment results meeting our criteria for potential miRNA:target mRNA interaction (475.0%in20–30nt overlap,470.0%in31–40nt overlap,465.0%in41–60nt overlap,460.0%in 61–80nt overlap,455.0%in81–100nt overlap and 440.0%in4101nt overlap).The proteins and inter-leukins were organized into three separate tables based on literature reports of their expression pattern and serum levels during HIV-1infection.
Table1lists proteins and interleukins(CD28,CD40L, IL-2,IL-3,TNF-b;IL-12and CD4)for which a strong consensus exists that the expression and serum levels are lower in HIV-1-positive patients in comparison to HIV-1-negative controls.We would expect according to our hypothesis that such proteins might be in?uenced by HIV-1derived miRNAs.Multiple alignments that matched the criteria were identi?ed for all proteins and interleukins in Table1.Table2lists proteins and interleukins(IL-10,IL-15,TNF-a;IL-6,IL-13and IL-18)for which reports are mixed concerning the expression pattern and serum levels in HIV-1-positive patients compared to HIV-1-negative controls.Align-ments in Table2that matched the criteria also were indicated for all proteins and interleukins in Table2. Finally,Table3lists proteins and interleukins(CTLA-4, IL-4,TGF-b1;IL-16and IL-1b)for which a strong consensus exists that the expression pattern and serum levels are higher in HIV-1-positive patients in compar-ison to HIV-1-negative controls.We would not expect there to be a signi?cant number of miRNA-like alignments between HIV-1and such proteins.The resulting alignments for CTLA-4,IL-4,TGF-b1and IL-1b are shown in Table3.These are clearly far less frequent than in Table1.No signi?cant alignments were found for IL-16.
An overall examination of the alignment results in Tables1–3relative to each other(i.e.many fewer regions of complementarity in Table3in comparison to Tables1and2)reveals an excellent correlation between the HIV-1vs.protein alignments and the reported decrease or increase in the expression pro?le of that protein.Of particular interest,are the many regions of RNA complementarity between HIV-1and CD28(Fig.
2),suggesting that the observed decrease in CD28 expression during HIV-1infection may be a result of miRNA activity.Fig.2shows the LFasta alignment results between the HIV-1proviral genome (nt7001–8000and9001–9748nt)and the complemen-tary sequence of CD28mRNA cds.The boxed nucleo-tide regions were considered to be potential miRNA:target mRNA binding sites based on the previously mentioned research?ndings regarding such interactions.
The hypothesis that these protein and interleukin mRNAs are subject to RNAi activity is further supported by a web-based siRNA design program (‘‘siDESIGN Center’’at https://www.360docs.net/doc/8c3025610.html,/ rnadesign/default.aspx)from Dharmacon Inc.(Rey-nolds et al.,2004).This program identi?ed siRNA target region sequences within the mRNA of all proteins and interleukins mentioned in this report(data not shown).Target sequences were speci?ed for the50UTR, ORF and30UTR of each mRNA cds,and many of these target regions corresponded exactly to sequences within the regions of complementarity indicated in Tables1–3. As it is well known that the major difference between HIV-1and HIV-2is their relative degree of pathogeni-city,we conducted alignments between HIV-2(EMBL-Bank database,AC X05291:Allison1994;Guyader et al.,1987;Clavel et al.,1986)and each protein/ interleukin in this report.The same methodology was employed(1000-nt windows of the HIV-2proviral genome vs.the entire mRNA cds of each protein and interleukin)and the same criteria for the identi?cation of signi?cant alignments were applied(475.0%in 20–30nt overlap,470.0%in31–40nt overlap, 465.0%in41–60nt overlap,460.0%in61–80nt
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overlap,455.0%in 81–100nt overlap and 440.0%in 4101nt overlap).The results are shown in Table 4and indicate much fewer regions of complementarity be-tween HIV-2and the proteins examined.The overall disparity in the results between HIV-1and HIV-2becomes more evident when comparing the results in Table 4in its entirety to the results in Tables 1–3.This disparity between HIV-1and HIV-2is not too unexpected when the low homology between the HIV-1and HIV-2genomes is considered (Guyader et al.,1987;Clavel et al.,1986).Assuming both HIV-1and HIV-2to be potential sources of miRNA,these results suggest a possible explanation for the differences in pathogenicity between HIV-1and HIV-2.
4.Discussion
We are not the ?rst to explore possible roles for RNAi in AIDS.Substantial research describes how RNAi mechanisms interfere with HIVreplication and how these processes could be exploited for therapeutic strategies against HIV(Dave and Pomerantz,2003;Stevenson,2003;Capodici et al.,2002;Veres et al.,1996).The extent of replicative control of HIVis
determined by a variety of factors such as the size of RNA,target sequence,RNA expression level and whether the RNA is sense or antisense to its target (Veres et al.,1996;Joshi et al.,1991).Also,expression from various regions within the genome (i.e.Tat,Nef,Vif or Rev)and transcription during particular points of the viral life cycle (i.e.prior to reverse transcription or after viral transcription from the integrated provirus)can be disrupted (Coburn and Cullen,2002;Jacque et al.,2002;Lee et al.,2002;Novina et al.,2002).Methods have also been developed that involve intro-ducing exogenous siRNA that targets the host cellular genes for the CD4or CCR5receptors,subsequently interfering with the membrane fusion and infection of a cell by HIV(Cullen,2002;Lehner,2002;Novina et al.,2002).
Interestingly,while much research has been con-ducted describing the effects of RNAi against HIV replication,the possibility that HIVcould exploit RNAi as a signi?cant mode of pathogenesis against host cells has not been considered.The data presented here suggest that HIV-1may use an RNAi mechanism to interfere with many T-cell functions.This would not come as a surprise as dsRNA (often a precursor of siRNA and miRNA)is present in cells infected with
Table 4
Alignment identities (i.e.regions of complementarity)between the HIV-2proviral genome and CD4+T-cell and macrophage protein/cytokine mRNA cds Protein/interleukin Identities
Protein/interleukin Identities
Protein/interleukin Identities
CD2865.2%in 46nt overlap IL-1085.7%in 21nt overlap CTLA-4
46.2%in 240nt overlap 77.3%in 22nt overlap CD40L
56.8%in 125nt overlap IL-1576.0%in 25nt overlap 81.0%in 21nt overlap 71.4%in 49nt overlap 76.2%in 21nt overlap 66.0%in 47nt overlap TNF-a 81.0%in 21nt overlap 80.0%in 20nt overlap 72.2%in 36nt overlap 78.6%in 28nt overlap IL-6
44.2%in 163nt overlap IL-475.0%in 24nt overlap 77.3%
in 22nt
overlap
57.5%in 120nt overlap 71.0%in 31nt overlap TGF-b 1
57.3%in 89nt overlap IL-2
54.7%in 128nt overlap 75.0%in 24nt overlap
83.3%in 24nt overlap 67.3%in 55nt overlap 71.8%in 39nt overlap IL-1343.8%in 502nt overlap IL-16None
IL-371.4%in 28nt overlap IL-18
None
IL-1b 75.0%in 20nt overlap
TNF-b 56.5%in 124nt overlap IL-12a chain 75.0%in 20nt overlap IL-12b chain 67.2%in 64nt overlap 71.0%in 31nt overlap CD4
79.2%in 24nt overlap 77.3%in 22nt overlap 75.0%in 20nt overlap
The listed identities are those for which 1000-nt windows of the HIV-1proviral genome were aligned against the entire mRNA cds of each protein (see text for description).An overall inspection of the alignment results in Table 4relative to the alignment results in Tables 1–3suggest a strong correlation between the alignment results and the well-known differences in pathogenicity between HIV-1and HIV-2.
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viruses such as rubella virus and Semliki Forest virus (Lee et al.,1994).It is also well known that dsRNA intermediates are a signi?cant part of an RNA virus replication cycle.Other viruses known to synthesize dsRNA include Adenovirus,HSV,Vaccinia,Reovirus and Encephalomyocarditis virus(Dave and Pomerantz, 2003),all of which might produce siRNA or miRNA. The next logical task would involve searching for potential miRNA encoded by these other viruses,as demonstrated by Pfeffer and coworkers,and determin-ing the consequences to host cellular activities.The results we report here make such a search plausible. The reported regions of complementarity between HIV-1and the T-cell and macrophage proteins and cytokines(Tables1–3)suggest the possibility of HIV-1-derived miRNA:host cell mRNA interactions and the potential of HIV-1exploiting RNAi mechanisms as a mode of pathogenesis.Of particular note are the two relatively extensive regions of complementarity between HIV-1and CD28(Fig.2)which show gapped alignment results of46.8%in391nt overlap and44.9%in379nt overlap.As shown in Fig.2,within these extensive regions of complementarity were numerous individual segments of consecutive nt complementarity ranging from2to10nt,many of which were4–6nt.Similar regions of complementarity were also obtained between HIV-1and CD40L,IL-2,IL-3,TNF-b;IL-12and CD4. Current evidence indicates that translational suppres-sion can occur if numerous short regions of partial complementarity(of which these regions could act in a cooperative fashion)exist between the miRNA and its target mRNA(Novina et al.,2002;Doench et al., 2003;Zeng et al.,2002,2003;Hutvagner and Zamore, 2002).With the exception of CTLA-4in Table3, comparable regions of complementarity were not found between HIV-1and IL-4,TGF-b1;IL-16and IL-1b: Whether these correlations between putative miRNA sequences and the reports of decreased CD28and cytokine expression in HIV-1-positive patients provide a mechanism for explaining some aspects of AIDS pathogenesis obviously needs to be tested experimen-tally.
We found only one major challenge to our correlation between putative HIV-1-derived miRNAs and experi-mental observations of decreased receptor or cytokine production,and that involved CTLA-4(Table3).The region of complementarity between HIV-1and CTLA-4 (46.0%in235nt overlap)was unexpected as reports indicate higher CTLA-4expression in HIV-1-positive patients in comparison to HIV-1-negative controls (Leng et al.,2002;Steiner et al.,1999).A possible reason for this contradiction may involve the opposing effects that CD28and CTLA-4exhibit following T-cell activation(Egen et al.,2002;Alegre et al.,1998; Krummel and Allison,1995;Linsley and Ledbetter, 1993).Interference with CD28may raise CLTL-4expression even in the presence of miRNA against CTLA-4mRNA(Fig.1).
It is important to consider other factors that may offer explanations to the contradictions(i.e.,the region of complementarity between HIV-1and CTLA-4 against reports of increased CTLA-4expression in HIV-1-positive patients)to our hypothesis.Methods that have been developed to identify potential mRNA targets indicate that a single miRNA can have multiple mRNA targets(Kiriakidou et al.,2004;Lewis et al., 2003)or a target mRNA can be acted upon by multiple miRNA molecules simultaneously(Doench and Sharp, 2004).This obviously complicates the analysis of the meaning of any individual region of complementarity. Also,mRNA translational suppression can be affected by the speci?city of base pairing that occurs within the50 region of the miRNA(Doench and Sharp,2004).It may be that the regions of complementarity between HIV-1 and CTLA-4are simply non-functional.The kinetics of HIV-1replication and mutation also are other factors to consider in these events(Jetzt et al.,2000;Cof?n,1995; Ho et al.,1995;Drake,1993),since the rate and timing of the production of various HIV-1RNAs may signi?cantly affect the function of any putative miRNA sequence.These factors may also play a role in the confused picture that emerges concerning expression patterns and serum levels of IL-10,IL-15,TNF-a;IL-6, IL-13and IL-18during HIV-1infection with regard to our alignment results(Table2).
We emphasize that the reported alignments are those for which1000-nt windows of the HIV-1proviral genome were aligned as previously described.Of course, the alignment window could be altered in numerous ways,by length or by start and end points.However, until experimental validation of HIV-1encoded miRNA can be unequivocally veri?ed and until our knowledge of siRNA:target mRNA interactions becomes more crystallized,it would be meaningless to make extensive alterations to the HIV-1alignment window to conduct more alignments and further attempt to determine the implications.The remarkable correlation amongst the alignment results in this report,the currently established criteria for siRNA:target mRNA interaction,and the reported expression pattern of various proteins and interleukins during HIV-1infection raises the question of HIV-derived miRNA(and miRNA from potentially many other viruses)as being a major source of immunodysfunction.
Based on existing reports that describe the mechanics of RNAi and miRNA production(see Bartel,2004; Cullen,2002;Agrawal et al.,2003for review),some plausible events can be deduced if HIV-1were produ-cing miRNA.It is likely after cleavage by the DICER enzyme and complete maturation of the miRNA in the cytoplasm that the miRNA:mRNA interactions may become effective.Current models suggest the occurrence
J.P.Couturier,R.S.Root-Bernstein/Journal of Theoretical Biology235(2005)169–184179
of either mRNA degradation if the miRNA is perfectly complementary to its mRNA target(Hutvagner and Zamore,2002)or translational repression if partially complementary(Doench et al.,2003;Saxena et al.,2003; Zeng et al.,2002,2003;Hutvagner and Zamore,2002). Subsequent to effective miRNA inhibition of CD28and CD4translation,probable consequences of this inhibi-tion include impaired cytokine production,anergy and apoptosis,and inability to stimulate APCs and B cells. The production of other cytokines not mentioned in this report may also be indirectly affected by HIV-1-produced miRNA(Jackson et al.,2003).Based on our alignment?ndings,we believe that translational repres-sion by partially complementary miRNA is accounting to some extent for the decreased CD28and CD4 expression and the dysregulation of cytokine production in HIV-1-infected T cells.These potential miRNA:mR-NA interactions are based on current knowledge of miRNA biogenesis and mechanisms as elucidated mainly from non-viral entities,but as many of these mechanisms appear to be highly conserved among numerous unrelated organisms,it would be reasonable to assume these processes to be applicable to viruses as well when initiating a more extensive investigation into this issue.It is clear that the types of experiments performed by Pfeffer et al.(2004)will be required to determine whether the sequences identi?ed here as possible miRNA actually perform inhibitory functions in vivo.
Of one thing,we can be certain.The?ndings in this report demonstrate that there is an extraordinary interplay between the mechanisms of viral pathogenesis and the mechanisms of host cellular immunity involving nucleic acid complementarity between their genomic makeup.If the existence and effects of viral-encoded miRNA are proven to be a signi?cant mode of transcriptional and translational regulation by viruses such as HIV-1,this could inspire new insights, hypotheses and experimental approaches for interven-tion and therapy,such as development of exogenous RNAi to block viral RNAi.This could also lead to more explicit explanations of viral pathogenesis and viral latency that go beyond an understanding of cell surface proteins and receptors.Polynucleotide complementarity between virus and host cell interactions may produce interactions that have previously resisted explanation. We hope the?ndings of our nucleic acid sequence alignments offer new venues for investigation into the pathogenesis of HIVinfections and perhaps other pathogen–host interactions in general.
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