Epstein-Barr Virus Latency inBloodMononuclear Cells: Analysis ofViral GeneTranscription during Primary Infection andintheCarrier State

Epstein-Barr virus (EBV)candisplay different formsoflatent infection inB-cell lines invitro; however, the typesofinfection normally established bythevirusinvivoremainlargely unexplored. Herewe have approached this question byanalyzing thetypes ofviral RNAspresent inmononuclear cells freshly isolated fromthebloodof14infectious mononucleosis patients undergoing primary EBVinfection and6long-term virus carriers. Reverse transcription-PCR amplifications werecarried outwithapanelofoligonucleotide primers andprobes whichspecifically detect (i)theEBER1RNA commontoallformsoflatency, (ii) transcripts either fromtheCpandWp promoters generating allsixnuclear antigen (EBNA1, -2,-3A, -3B, -3C, -LP)mRNAsorfromtheFppromoter generating auniquely spliced EBNA1mRNA,(iii) thelatent membrane protein (LMP1and2A)mRNAs,and(iv) theBZLF1mRNA,animmediate-early markeroflytic cycle. Viral transcription ininfectious mononucleosis mononuclear cells (andintheB-cell-enriched fraction) regularly included thefull spectrum oflatent RNAsseenduring EBV-induced B-cell growth transformation invitro, i.e., EBER1,Cp/Wp-initiated EBNAmRNAs,andLMP1/LMP2mRNAs,intheabsence oflytic BZLF1transcripts. Inaddition, transcripts withthesplice pattern ofFp-initiated EBNA1mRNA,hitherto seenonlyinvivoin certain EBV-positive tumors, werefrequently detected. Inlong-term virus carriers, themononuclear cells were again positive forlatent (EBER1)andnegative forlytic (BZLF1) markers; Cp/Wp-initiated RNAswerenot detected inthese samples, butinseveral individuals itwaspossible toamplify bothFp-initiated EBNA1mRNA andLMP2AmRNA signals. We suggest (i) thatprimary infection isassociated withatransient virus-driven expansion oftheinfected B-cell poolthrough a programofvirus geneexpression likethatseeninin vitro-transformed cells and(ii) thatlong-term virus carriage isassociated withaswitch fromCp/WptoFp usageandthustoamorerestricted formoflatent protein expression thatmayrender theinfected cells less susceptible torecognition bythevirus-specific cytotoxic T-cell response. Epstein-Barr virus (EBV), agammaherpesvirus widespread inhumanpopulations, naturally infects twomajor target cell types invivo, pharyngeal epithelium andmatureBlymphocytes (25). Pharyngeal epithelial cells appear tobenaturally permissive, withvirus replication indifferentiating squamous cells leading totherelease ofinfectious virions into buccal fluid (10, 42). Incontrast, B lymphocytes harbor thevirus asanonproductive (latent) infection (28), anditisthis interaction which appears central tothephenomenon ofviral persistence (9, 52). Studies oninfectious mononucleosis (IM)patients showthat a large pooloflatently infected B cells isgenerated invivo during thecourse ofprimary infection (16, 20,47). Although this poolissubstantially reduced insize oncethehostcellmediated immuneresponse develops (32, 34), small numbers ofinfected Bcells persist intheblood andlymphoid tissues of virus carriers, detectable byvirtue oftheir ability togive rise to EBV-transformed lymphoblastoid cell lines (LCLs) byspontaneousoutgrowth invitro (28,53). An important objective, therefore, istoidentify thetypes ofEBV-B-cell interaction whichwill allow ontheonehandefficient colonization ofthe lymphoid system during primary infection andontheother handlong-term persistence ofinfected cells inthefaceof sustained hostimmuneresponses.