254 K.J. MACBETH AND J.L. PATTERSONTHE JOURNAL OF EXPERIMENTAL ZOOLOGY 282:254–260 (1998)

© 1998 WILEY-LISS, INC.

Overview of the Leishmaniavirus Endoribonucleaseand Functions of Other Endoribonucleases AffectingViral Gene Expression

KYLE J. MACBETH1 AND JEAN L. PATTERSON2*1Department of Microbiology and Molecular Genetics, Harvard MedicalSchool, and Division of Infectious Diseases, Children�s Hospital, Boston,Massachusetts 02115

2Department of Virology and Immunology, Southwest Foundation forBiomedical Research, San Antonio, Texas 78228

ABSTRACT Leishmaniaviruses (LRV) are double-stranded RNA viruses that persistently in-fect some strains of the protozoan parasite Leishmania. The identification of a short viral RNAtranscript led to our discovery of an endoribonuclease activity of the LRV capsid protein. Otherknown endoribonucleases serve a variety of diverse roles in the regulated balance of processingand degradation of both cellular and viral RNAs, thus determining the amount and functionalityof specific RNA molecules in a cell at any given time. The consequence of LRV RNA cleavage onthe LRV life cycle has not yet been determined. Here we review the LRV endoribonuclease anddiscuss potential roles for this endoribonuclease activity in the context of the involvement of otherendoribonucleases in regulating viral gene expression and replicative capacity. J. Exp. Zool. 282:254�260, 1998. © 1998 Wiley-Liss, Inc.

LEISHMANIAVIRUS GENOMEORGANIZATION

Leishmaniaviruses (LRV) are members of thefamily Totiviridae that persistently infect somestrains of the protozoan parasite Leishmania(Patterson, ’93). LRV have been identified in 12isolates of the New World species Leishmaniaguyanensis and L. braziliensis, and one strain ofthe Old World species L. major (Guilbride et al.,’92; Cadd et al., ’93). Full-length cDNA clones havebeen generated for two New World isolates, LRV1-1 and LRV1-4, and one Old World isolate, LRV2-1(Stuart et al., ’92; Scheffter et al., ’94, ’95). TheLRV genome is approximately 5.3 kbp of double-stranded RNA (dsRNA), and two large open read-ing frames (ORF2 and ORF3) are present in allisolates. When ORF2 is expressed by recombinantbaculovirus in Spodoptera frugiperda cells, the 82-kDa protein self-assembles into virus-like particlesof identical morphology to native virions, demon-strating that ORF2 encodes the major capsid pro-tein (Cadd and Patterson, ’94). ORF 3 possessesmotifs characteristic of viral RNA-dependent RNApolymerases, and purified LRV virions possess bothtranscriptase and replicase activities that gener-ate positive- and negative-sense genome-lengthRNAs, respectively (Widmer et al., ’90; Widmer and

Patterson, ’91). Viral replication proceeds throughthe overproduction of a positive-sense RNA thatserves as both the viral mRNA and replicative in-termediate (reviewed in Wickner, ’93).

Together, ORFs 2 and 3 encompass over 90%of LRV genomes, leaving approximately 450nucleotides (nt) at the 5´ termini of LRV1 iso-lates and 340 nt at the 5´ terminus of LRV2-1.Although small ORFs can be identified in these5´ terminal sequences, the ORFs are not con-served among isolates and have not been shownto encode gene products. The nucleotide se-quences at the 5´ ends of LRV1 and LRV2 iso-lates are highly divergent, with less than 40%homology (Scheffter et al., ’95). However, thenucleotide sequence at the 5´ end of LRV1 iso-lates is highly conserved (90% identity), thoughthe remaining sequence exhibits divergence inthe form of third-base wobble (75% identity).

Grant sponsor: NIH NIAID; Grant number: 7R01 AI28473-04.Kyle J. MacBeth’s present address: Millennium Pharmaceuticals,

Inc., 640 Memorial Drive, Cambridge, MA 02139.*Correspondence to: Jean L. Patterson, Department of Virology

and Immunology, Southwest Foundation for Biomedical Research,P.O. Box 760549, San Antonio, Texas 78245-0549. E-mail: jpatters@icarus.sfbr.org

Received 27 March 1998; Accepted 27 March 1998

LEISHMANIAVIRUS ENDORIBONUCLEASE 255

Strong conservation of nucleotide sequence at the5´ ends of LRV1 genomes suggests that this re-gion serves an essential viral function.

IDENTIFICATION OF ALEISHMANIAVIRUS

ENDORIBONUCLEASE ACTIVITYBoth genome-length RNA and a short RNA

transcript are generated by LRV virions in an invitro polymerase assay (Widmer et al., ’89, ’90;Widmer and Patterson, ’91; Weeks et al., ’92a,b;Chung et al., ’94). The short transcript is anssRNA of approximately 320 nt in length, corre-sponding to the 5´ end of positive-sense viral RNA.Northern analysis of total RNA isolated from vi-rus-infected Leishmania cells showed that theshort viral RNA was also synthesized in vivo.With no known function for the 5´ terminal nucle-otides upstream of the initiating codon for the vi-ral capsid protein, it was intriguing that the shorttranscript represented positive-sense RNA fromthis region. It was originally hypothesized thatgeneration of the short transcript was mediatedvia premature transcription termination of theviral polymerase, but the possibility of a specificcleavage event had not been ruled out at thattime. In an attempt to distinguish between thesetwo mechanisms, the cleavage hypothesis wastested by searching for the downstream cleavageproduct counterpart predicted for an endoribo-nucleolytic cleavage event and by the developmentof an in vitro cleavage assay.

In an effort to identify the 5´ end of the pre-dicted downstream cleavage product, primer ex-tension analysis was performed on the RNAproducts from a polymerase assay of sucrose gra-dient-purified particles (MacBeth and Patterson,’95a). Using a primer that bound downstream ofthe putative cleavage site, a distinct primer ex-tension product was observed with a size map-ping to position 320 of the LRV1-4 genome.Because the LRV1-4 short transcript was approxi-mately 320 nt in length, the primer extensionexperiment suggested that the 5´ end of the down-stream cleavage product may have been identi-fied. This finding was the first indication that anRNA cleavage event may be occurring.

To determine more definitively whether theshort transcript was generated by a cleavageevent, an in vitro cleavage assay was developed(MacBeth and Patterson, ’95a). In vitro-synthe-sized substrate RNAs, possessing the 5´-un-translated region (UTR) of LRV1-4 positive-senseRNA, were incubated with sucrose gradient-puri-

fied native viral particles, and cleavage was as-sessed by resolving reaction products on a dena-turing polyacrylamide gel. In the cleavage assay,substrate RNAs were specifically cleaved into twodistinct products only in the presence of viral par-ticles, indicating that a factor(s) present in theviral particle preparation was mediating endo-nucleolytic cleavage of the RNAs. The endori-bonuclease activity was specific to substratespossessing the viral sequence, as a nonviral RNAwas not cleaved in the assay. The combinedlengths of the cleavage products equaled that ofthe input transcript, and the site of cleavage wasmapped to nucleotide 320 of the LRV1-4 genome.Nucleotide 320 was exactly the same position thatwas mapped by primer extension to the 5´ end ofthe predicted downstream cleavage product coun-terpart to the short transcript. In addition, thecleavage activity associated with viral particleswas directly associated with the ability of viralparticles to generate the short transcript in invitro polymerase assays, because viral particlesthat were unable to generate the short transcriptin polymerase assays did not function in cleavageassays. These data indicated that the short tran-script was generated by a site-specific endoribo-nucleolytic cleavage event within the 5´-UTR ofviral positive-sense RNA.

A LEISHMANIAVIRUS CAPSIDENDORIBONUCLEASE

The development of an in vitro cleavage assayfor the endoribonuclease activity associated withpurified Leishmaniavirus virions provided asystem in which to study both the enzyme andthe substrate of the cleavage reaction. MutantRNA substrates could be generated and tested forcleavage susceptibility in order to define RNA de-terminants of the endoribonucleolytic activity. Fur-thermore, the wild-type RNA substrate could beused to assay for the endoribonuclease activity ofvarious proteins in order to identify the componentof the native virion preparations that possessedthe endoribonuclease activity. The endoribo-nuclease responsible for the observed cleavage ac-tivity could have been of host or viral origin.Cleavage assays were initially performed usingnative virions, purified by sucrose gradient frac-tionation of virus-infected Leishmania cell ex-tracts, as the source of endoribonuclease activity.The possibility that a co-sedimenting or virallyincorporated host cellular factor was responsiblefor the endoribonuclease activity had not beenruled out. Expression of LRV capsid proteins in

256 K.J. MACBETH AND J.L. PATTERSON

recombinant-baculovirus expression systemsyields self-assembled viruslike particles of mor-phology identical to that of native virions. Hav-ing the LRV capsid protein expressed in a systemother than the natural Leishmania host providedthe reagent to test directly for an endoribonucleaseactivity of the viral capsid protein. When substrateRNAs were incubated with sucrose gradient-pu-rified recombinant-baculovirus-expressed LRVcapsid protein, the RNAs were specifically cleaved,generating cleavage products identical to thoseproduced upon incubation of the substrate RNAwith native viral particles (MacBeth and Patter-son, ’95b). The ability of the LRV capsid protein,expressed in a heterologous system, to functionin the cleavage assay identified the LRV capsidprotein as an endoribonuclease.

LEISHMANIAVIRUS CLEAVAGE SITESEQUENCES

Short RNA transcripts are generated in poly-merase assays of all LRV isolates tested, suggest-ing that the endoribonuclease activity is conservedin the entire LRV genus (MacBeth and Patterson,’95b). The endoribonuclease activity first demon-strated with an LRV1-4 capsid/substrate systemin vitro could be reproduced with an LRV2-1capsid/substrate system (MacBeth et al., ’97). Al-though the sequence of Old World virus LRV2-1is significantly divergent from the sequences ofthe New World LRV1 isolates (<50% homology), asequence in the 5´UTR of LRV2-1 is homologousto the cleavage site sequence in LRV1-4 RNA(MacBeth and Patterson, ’95b; Scheffter et al., ’95).To determine whether cleavage of LRV2-1 RNAoccurs within the region of sequence homology toLRV1 isolates, we accurately mapped the cleav-age site of LRV2-1 RNA by sizing the 5´ cleavageproduct generated in an in vitro cleavage assay.Using native LRV2-1 virions and a 5´-end-labeledLRV2-1-derived substrate RNA, a single cleavageproduct was generated with a size that mappedthe cleavage site to nucleotide 283 of the LRV2-1genome. Cleavage at this site is within the shortregion of sequence homology between the LRV2and LRV1 RNAs. The cleavage site in LRV1-4RNA is after viral nucleotide 320, between the twocytosine residues of the sequence 5´GAUC*CG-AA3´. The cleavage site in LRV2-1 RNA is afterviral nucleotide 283, immediately 5´ of the twocytosine residues of the sequence 5´GCU*CC-GUA3´. Although the cleavage sites in these twoviral RNAs did not map to exactly the same posi-tion within the consensus sequence 5´GxUC-

CGxA3´, the sites mapped to within one nucle-otide of each other. This one nucleotide discrep-ancy may represent a true difference in the siteof cleavage for each virus, or it may be attributedto an experimental limitation.

To determine whether the consensus sequencewas required for specific RNA cleavage, we gen-erated an RNA substrate with a 6-nucleotidedeletion in the consensus sequence and assayedfor cleavage by viral particles (MacBeth andPatterson, ’95b). The deletion mutant RNA sub-strate was not cleaved by viral particles, indicat-ing that cleavage requires the deleted consensussequence. The functional requirement and se-quence conservation in divergent viral isolatessuggests that this element is an important com-ponent of the determinant for targeting the spe-cific endoribonucleolytic cleavage in LRV RNAs.

Endoribonucleases can be divided into twoclasses on the basis of their RNA cleavage sitespecificities. Nonspecific endoribonucleases cleaveRNA at a large number of sites, although theseRNases often show a specificity for a particularbase at the site of cleavage (i.e., Aspergillus RNaseT1 cleaves after any guanosine residue). In con-trast, the highly specific endoribonucleases havemore complex cleavage site determinants and lim-ited substrate specificities. The mechanism ofcleavage site selection for the specific endori-bonucleases remains an enigma, despite extensiveattempts to delineate the required determinantsin substrate RNA molecules. Targeting the site ofcleavage in an RNA could be mediated by primarynucleotide sequences, secondary/tertiary struc-tures, or a combination of both. In almost all stud-ies aimed at identifying the RNA cleavage sitedeterminants of specific endoribonucleases, no pre-cisely conserved sequence element has been iden-tified that is required for specific RNA cleavageto occur (Apirion and Miczak, ’93).

To determine whether the putative consensussequence for RNA cleavage in a New WorldLeishmaniavirus RNA could target cleavage by anOld World Leishmaniavirus capsid endoribo-nuclease, and vice versa, cleavage assays were per-formed with LRV2-1 and LRV1-4 virions on bothcognate and noncognate substrate RNAs (Mac-Beth et al., ’97). The LRV2-1 capsid endoribonu-clease preferentially cleaved the cognate substrateRNA with 35-fold better efficiency than thenoncognate substrate RNA, although the non-cognate substrate RNA was cleaved at a low levelat the wild-type cleavage site. The LRV1-4 capsidendoribonuclease did not demonstrate any signifi-

LEISHMANIAVIRUS ENDORIBONUCLEASE 257

cant substrate specificity with regard to cleavageefficiency, although the noncognate substrate RNAwas cleaved at a site approximately 10 nucleotides5´ to the wild-type cleavage site. Therefore, theRNA determinants that dictate the site-specifictargeting of the LRV capsid endoribonuclease arelikely to include both primary nucleotide se-quences and higher order structures. Although aputative consensus sequence for cleavage was pro-posed for the LRV capsid endoribonuclease, thedifferential cleavage efficiencies of different sub-strates that possess this consensus sequence sug-gest that other determinants are involved.

GENERAL FEATURES OFENDORIBONUCLEASES

Endoribonucleases serve diverse roles in thegeneration of functional RNAs, the activation ofexpression from RNAs, and the physical and func-tional inactivation of RNAs (Belasco, ’93; Court,’93). RNA processing can be described as thoseevents, following transcription, which make anRNA molecule functional. Endoribonucleolyticcleavages represent one type of RNA processingreaction. Other examples of RNA processing re-actions include exoribonucleolytic end-trimming,RNA splicing, RNA editing, and addition of nucle-otides to RNA termini (Apirion and Miczak, ’93).RNA synthesis and processing activities arecomplemented by RNA degrading activities, de-scribed as those reactions that mediate the de-struction, and therefore functional removal, ofRNA molecules. RNA degradative activities areprimarily mediated through exoribonucleolyticcleavages, although endoribonucleolytic cleavageevents are becoming increasingly evident in theinitiating rate-limiting step of some degradativepathways (Stevens, ’93; Decker and Parker, ’94;Beelman and Parker, ’95). The regulated balanceof transcription, processing, and degradation de-termines the level and functionality of RNA mol-ecules in a cell at any given time (Apirion andMiczak, ’93).

ENDORIBONUCLEASE FUNCTIONSIN PHAGE SYSTEMS

Perhaps most intriguing with regard to anybiological significance of an endoribonuclease ac-tivity in the Leishmaniavirus system is the pre-cedent in other viral systems for a functional roleof a site-specific endoribonucleolytic cleavageevent in the 5´UTR of viral RNAs. In phagelambda and phage T7 systems, RNase III-medi-ated endoribonucleolytic cleavage in the 5´UTRs

of specific early viral RNAs serves to activatetranslation of downstream ORFs by removingtranslation attenuating secondary structures thatinhibit ribosome binding at translation initiatingsequences (Court, ’93). In T4-related phages,endoribonucleolytic cleavage in the 5´UTR of vi-ral RNAs serves to inactivate translation by re-moving sequences required for ribosome binding(Sanson and Uzan, ’95). Regulated expression ofearly viral RNAs in response to a cellular modu-lator of RNA expression could serve to control vi-ral replication in concert with varying cellularconditions. Although phages lambda and T7 uti-lize a host endoribonuclease to regulate viral geneexpression, phage T4 encodes its own regulatoryendoribonuclease, Reg B. Reg B endoribonucleasecleaves at predominantly sequence-specific sitespresent in the Shine-Dalgarno sequences of earlyviral messages, serving to turn off gene expres-sion irreversibly by functionally inactivatingcleaved mRNAs. A Reg B cleavage site is alsopresent in its own mRNA, and Reg B expressionhas been shown to be auto-regulated as a func-tion of cleavage at this site (Sanson and Uzan,’95). This cleavage event tightly regulates geneexpression at the level of translation inhibition,by removing sequences required for ribosomebinding, and at the level of decreased messagestability, presumably by removing structures thatprevent message degradation.

LEISHMANIAVIRUSENDORIBONUCLEASE FUNCTIONS,

PERSISTENT INFECTION, AND AREPLICATION MODEL

Endoribonucleolytic cleavage within the 5´UTRof LRV RNA is mediated by the capsid end-oribonuclease encoded by the ORF immediatelydownstream of the cleavage site. In analogy to theReg B system, it is tempting to speculate that thepersistent nature of LRV infection is auto-regu-lated as a function of a capsid-mediated endori-bonucleolytic cleavage event that serves to affectthe translational efficiency of viral RNA. A recentstudy presented evidence that the 5´UTR of LRVcan function as an internal ribosome entry sitefor cap-independent translation in Leishmania(Maga et al., ’95). Furthermore, a 120 nt deletionin the region that is removed by cleavage wasshown to reduce translation by 10-fold. Regulat-ing viral translation below a specific thresholdlevel is the mechanism utilized by coronavirusesto maintain a persistent infection (Hofmann et al.,’93). The conservation of a capsid endoribonuclease

258 K.J. MACBETH AND J.L. PATTERSON

activity in divergent LRV isolates, infecting dif-ferent species of Leishmania, suggests that viralRNA cleavage is likely to serve an important rolein the maintenance of LRV persistence.

Infection of Leishmania cells by LRV is be-lieved to be persistent, as no extracellular par-ticles have ever been observed and isolatedvirions cannot stably infect virus-free Leishma-nia (Croft and Molyneux, ’79 reviewed in Patter-son, ’93; Armstrong et al., ’93). Furthermore,Leishmania reproduction is considered to be pre-dominantly asexual (Tibayrenc et al., ’90, ’91;Panton et al., ’91). The lack of an extracellularinfectious cycle for the virus, combined with thelack of sexual reproduction for the host para-site, would result in co-evolution of virus andparasite in divergent lineages such that no ge-netic recombination of LRV would occur. With-out recombination, evolution of LRV genomeswould be expected to diverge in parallel with thespeciation of the ancestral host parasite. Indeed,a phylogenetic comparison of LRV genomic se-quences with DNA fingerprints from the hostparasite strains showed a similar relationshipamong virus and parasite genetic distances (Wid-mer and Dooley, ’95). This analysis suggests thateach strain of LRV has co-existed in long-termassociation with each respective Leishmaniastrain.

The exact functional role in viral replicationfor the LRV capsid endonuclease activity re-mains to be determined. In addition to possibletranslational effects, RNA cleavage is likely toaffect the turnover rate of viral RNA. Althoughthe RNA products of an endoribonucleolyticcleavage event may have increased or decreasedstabilities relative to the uncleaved transcript,our cleavage assays suggest that the downstreamLRV RNA is destabilized by cleavage, as the 3´cleavage product of LRV-derived substrate RNAsrapidly disappears, while the 5´ cleavage prod-uct persists. The combination of translationaland stability effects of viral RNA cleavage wouldaffect the viral replication level by determiningthe amount of viral proteins and template RNAavailable for virus production.

A model for the replication cycle of LRV is dia-grammed in Figure 1. Cleaving the 5´ end frompositive-sense, genome-length viral transcriptscould serve to create a subset of viral RNAs thatare translation and replication incompetent. Ob-viously a virus cannot destroy all of its functionalmRNA and continue to replicate. Our model pre-dicts that, at low virus densities (i.e., during log-

phase growth of the Leishmania), a portion ofgenome-length transcripts will avoid cleavage, betranslated, and subsequently be encapsidated. Asthe density of virus in the cell accumulates (i.e.,during the stationary phase of the Leishmania),all viral transcripts will encounter a capsid en-donuclease and be cleaved. Such a suicide mecha-nism would serve to regulate tightly the numberof virions per cell at a low level, thereby main-taining a persistent infection. Consistent with thismodel, recent work characterizing the productionof viral transcripts throughout the growth curveof in vitro-cultured Leishmania cells demonstratesthat, as parasites progress from log-phase growthto stationary-phase growth, the relative abundanceof short transcripts to full-length positive-sensetranscripts increases (Chung et al., unpublishedobservation).

Alternative roles for the endonuclease activityexist. Cleavage of viral transcripts could serve togenerate functional mRNAs by removing poten-tial translation attenuating domains present atthe 5´ end of full-length RNAs. Furthermore, thesmall ORFs present in the 5´ ends of LRV genomesare intact in the short transcript generated bycleavage, and, therefore, the possibility that theshort transcript may serve as an mRNA for thesegene products cannot be discounted. In addition,the cytoplasmic subcellular localization of LRV(Tarr et al., ’88; Cadd and Patterson, ’94) suggeststhat both viral and cellular RNAs present in thecytoplasm could be accessible to the capsid endo-nuclease activity. Differences in phenotypes be-tween uninfected and virus-infected Leishmania

Fig. 1. A model for the replication cycle of Leishmaniaviruses.

LEISHMANIAVIRUS ENDORIBONUCLEASE 259

could reflect differences in expression of cleavage-susceptible mRNAs.

The identification of an endoribonucleaseactivity of the LRV capsid protein is unprec-edented for viral capsids. The capsid proteins ofdsRNA viruses appear to possess structurallyunique properties that are likely attributed to thefact that, unlike all other classes of viruses,dsRNA viruses utilize their capsids as compart-ments for the active synthesis of viral nucleic ac-ids (Cheng et al., ’94). It will be interesting tosee if the other Totiviridae possess similar capsid-encoded endoribonuclease activities. The identi-fication of a novel enzymatic activity of a viralcapsid protein exemplifies the ability of virusesto attain maximal function from minimal codingcapacity and expands the possible strategicmechanisms viruses may utilize for obligate in-tracellular existence.

LITERATURE CITEDApirion, D., and A. Miczak (1993) RNA processing in prokary-

otic cells. Bioessays, 15:113–120.Armstrong, T.C., M.C. Keenan, G. Widmer, and J.L. Patterson

(1993) Successful transient introduction of Leishmania RNAvirus into a virally infected and an uninfected strain ofLeishmania. Proc. Natl. Acad. Sci. USA, 90:1736–1740.

Beelman, C.A., and R. Parker (1995) Degradation of mRNAin eukaryotes. Cell, 81:179–183.

Belasco, J.G. (1993) mRNA degradation in prokaryotic cells:an overview. In: Control of Messenger RNA Stability. J.Belasco and G. Brawerman, eds. Academic Press, San Di-ego, pp. 3–12.

Cadd, T.L., and J.L. Patterson (1994) Synthesis of viruslikeparticles by expression of the putative capsid protein ofLeishmania RNA virus in a recombinant baculovirus ex-pression system. J. Virol., 68:358–365.

Cadd, T.L., M.C. Keenan, and J.L. Patterson (1993) Detec-tion of Leishmania RNA virus 1 proteins. J. Virol., 67:5647–5650.

Cheng, R.H., J.R. Caston, G.-J. Wang, F. Gu, T.J. Smith, T.S.Baker, R.F. Bozarth, B.L. Trus, N. Cheng, R.B. Wickner,and A.C. Steven (1994) Fungal virus capsids, cytoplasmiccompartments for the replication of double-stranded RNA,formed as icosahedral shells of asymmetric gag dimers. J.Mol. Biol., 244:255–258.

Chung, I.K., T.C. Armstrong, and J.L. Patterson (1994) Iden-tification of a short viral transcript in Leishmania RNA vi-rus-infected cells. Virology, 198:552–556.

Court, D. (1993) RNA processing and degradation by RNaseIII. In: Control of Messenger RNA Stability. J. Belasco andG. Brawerman, eds. Academic Press, San Diego, pp. 71–116.

Croft, S.L., and D.H. Molyneux (1979) Studies on the ultra-structure, virus-like particles and infectivity of Leishma-nia hertigi. Ann. Trop. Med. Parasitol., 73:213–226.

Decker, C.J., and R. Parker (1994) Mechanisms of mRNA deg-radation in eukaryotes. Trends Biochem. Sci., 19:336–340.

Guilbride, L., P.J. Mylar, and K. Stuart (1992) Distributionand sequence divergence of LRV1 viruses among differentLeishmania species. Mol. Biochem. Parasitol., 54:101–104.

Hofmann, M.A., S.D. Senanayake, and D.A. Brian (1993) Atranslation-attenuating intraleader open reading frame isselected on coronavirus mRNAs during persistent infection.Proc. Natl. Acad. Sci. USA, 90:11733–11737.

MacBeth, K.J., and J.L. Patterson (1995a) The short tran-script of Leishmania RNA virus is generated by RNA cleav-age. J. Virol., 69:3458–3464.

MacBeth, K.J., and J.L. Patterson (1995b) Single-site cleav-age in the 5´-untranslated region of Leishmaniavirus RNAis mediated by the viral capsid protein. Proc. Natl. Acad.Sci. USA, 92:8994–8998.

MacBeth, K.J., Y-T. Ro, L. Gehrke, and J.L. Patterson (1997)Cleavage site mapping and substrate-specificity of Leish-maniavirus 2-1 capsid endoribonuclease activity. J. Bio-chem., 122:193–200.

Maga, J.A., G. Widmer, and J.L. Lebowitz (1995) Leishma-nia RNA virus 1-mediated cap-independent translation. Mol.Cell. Biol., 15:4884–4889.

Panton, L.J., R.B. Tesh, K.C. Nadeau, and S.M. Beverley(1991) A test for genetic exchange in mixed infections ofLeishmania major in the sand fly Phlebotomus papatasi. J.Protozool., 38:224–228.

Patterson, J.L. (1993) The current status of Leishmania vi-rus 1. Parasitol. Today, 9:135–136.

Sanson, B., and M. Uzan (1995) Post-transcriptional controlsin bacteriophage T4: roles of the sequence-specific endori-bonuclease RegB. FEMS Microbiol. Rev., 17:141–150.

Scheffter, S., G. Widmer, and J.L. Patterson (1994) Completesequence of Leishmania RNA virus 1-4 and identificationof conserved sequences. Virology, 199:479–483.

Scheffter, S., Y.-T. Ro., I.-K. Chung, and J.L. Patterson (1995)The complete sequence of Leishmania RNA virus LRV2-1, avirus of an Old World parasite strain. Virology, 212:84–90.

Stevens, A. (1993) Eukaryotic nucleases and mRNA turn-over. In: Control of Messenger RNA Stability. J. Belascoand G. Brawerman, eds. Academic Press, San Diego, pp.449–471.

Stuart, K.D., R. Weeks, L. Guilbride, and P.J. Mylar (1992)Molecular organization of Leishmania RNA virus 1. Proc.Natl. Acad. Sci. USA, 89:8596–8600.

Tarr, P.I., R.F. Aline, B.L. Smiley, J. Scholler, J. Keithly, andK. Stuart (1988) LR1: A candidate RNA virus of Leishma-nia. Proc. Natl. Acad. Sci. USA, 85:9572–9575.

Tibayrenc, M., F. Kjellberg, and F.J. Ayala (1990) A clonaltheory of parasitic protozoa: the population structures ofEntamoeba, Giardia, Leishmania, Naegleria, Plasmodium,Trichomonas, and Trypanosoma and their medical and taxo-nomical consequences. Proc. Natl. Acad. Sci. USA, 87:2414–2418.

Tibayrenc, M., F. Kjellberg, J. Arnaud, B. Oury, S.F. Breniere,M.-L. Darde, and F.J. Ayala (1991) Are eukaryotic microor-ganisms clonal or sexual? A population genetics vantage.Proc. Natl. Acad. Sci. USA, 88:5129–5133.

Weeks, R., R.F. Aline, P.J. Myler, and K. Stuart (1992a)LRV1 viral particles in Leishmania guyanensis containdouble-stranded or single-stranded RNA. J. Virol., 66:1389–1393.

Weeks, R.S., J. L. Patterson, K. Stuart, and G. Widmer (1992b)Transcribing and replicating particles in a double-strandedRNA virus from Leishmania. Mol. Biochem. Parasitol.,52:207–214.

Wickner, R.B. (1993) Double-stranded RNA virus replicationand packaging. J. Biol. Chem., 268:3797–3800.

Widmer, G., and S. Dooley (1995) Phylogenetic analysis ofLeishmania RNA virus and Leishmania suggests ancient

260 K.J. MACBETH AND J.L. PATTERSON

virus-parasite association. Nucleic Acids Res., 23:2300–2304.

Widmer, G., and J.L. Patterson (1991) Genomic structure andRNA polymerase activity in Leishmania virus. J. Virol.,65:4211–4215.

Widmer, G., A.M. Comeau, D.B. Furlong, D.F. Wirth, and J.L.

Patterson (1989) Characterization of a RNA virus from theparasite Leishmania. Proc. Natl. Acad. Sci. USA, 86:5979–5982.

Widmer, G., M.C. Keenan, and J.L. Patterson (1990) RNApolymerase activity is associated with viral particles iso-lated from Leishmania braziliensis subsp. guyanensis. J.Virol., 64:3712–3715.