Bacterial 3-Galactosidase asaMarker ofRousSarcoma Virus Gene Expression andReplication

Wehavedeveloped aconvenient andsensitive assay ofeucaryotic geneexpression which usestheEscherichia coli lacZgeneproduct, j-galactosidase, asanonselectable marker. Thissystem hasbeenapplied totheanalysis ofRoussarcoma virus replication andgeneexpression. Aviancells weretransfected withplasmids encoding in-frame genefusions oftheN-terminal portion ofthegaggenetoa 'lacZ gene, whichrequires both transcriptional andtranslational initiation signals; these weresupplied bythevirus long terminal repeat and leader region. Readily detectable quantities of0-galactosidase weresynthesized intransfected cells; itwas demonstrated that thelevels ofenzyme activity induced insuchcultures increased linearly withtheinput DNA concentration andalso correlated withmRNA levels. Byusing aRoussarcomavirus-derived vector containing thesrcgeneandarelated virus asahelper, itwasshownthat lacsequences werecompatible withallphases ofthevirus life cycle. gag-lacZ fusion proteins wereimmunoprecipitable fromcultures whichstably expressed lacZaswell assrc. Virus rescued fromstably transfected cultures resulted incontinued lacandsrcexpression inrecipient cells. Oneparticular construction wasefficiently transmitted asvirus, although itlacked sequences thought tobeimportant forencapsidation ofRNAinto virions. Thedatapresented heredemonstrate theuse oflacZasamarkerofretrovirus geneexpression andreplication. Retrovirus replication andgeneexpression areinterwoven inacomplex fashion, asarethesequences whichregulate these processes. Asaconsequence, anunderstanding ofthe viral genomerequires notonlydissection ofisolated functional regions, butalsoanalysis ofthemannerinwhich different regions interact, bothstructurally andfunctionally. Studies ofdeletion mutants haveshownthattheproducts encoded bytheviral structural genesofgag,pol, andenv maybesupplied intrans, andhaveidentified certain regions ofthegenomewhichactincis. Regions required incisfor reverse transcription ofgenomeRNA,integration ofproviralDNA,andsynthesis ofRNA include thelongterminal repeats (LTRs) andadjacent noncoding regions (45). The LTRswhichflank anintegrated DNA provirus arecomposed ofsequences derived fromboththe5'and3'endsof theRNAgenome(refered toasU5andU3,respectively) and contain signals fortranscription initiation andforgeneration of3'endsofviral genomeandmRNA.Itispresumed that thegenomecontains sequences whichmodulate thesplicing ofmRNAs,asnotall viral transcripts arespliced; still other sequences direct theencapsidation offull-length RNA genomes. Thereisgenetic evidence that asequence within the untranslated leader region nearthe5'endofthegenomeis required forpackaging ofRoussarcoma virus (RSV)genomesinto virions (22, 39). A similar function hasalso been attributed to115bases nearthe3'endofthegenome(44), as wellastoa 28-base near-perfect palindrome whichlies within thegagcoding sequence (35). However, itisdifficult toassign specific functions tothese regions, asanymutation might havepleiotropic effects. Although theexpression ofvirtually anygene,viral or otherwise, canbemonitored bydirectly measuring the accumulation ofRNA transcripts after introduction into an appropriate cell type, itisoften simpler tosubstitute an indicator genewhichconfers areadily detectable phenotype.