The long range goals of this project are to understand molecular mechanisms which control lentivirus replication and expression in vivo.
The specific aims are to functionally characterize variable regions in the LTR and rev gene of EIAV. The biological significance of variation in the LTR will be studied in transient expression assays using CAT (chloramphenicol acetyl transferase) as a reporter gene. To determine if variability in the LTR plays a role in cell tropism, the investigator proposes performing assays in cells previously shown to differ in permissiveness for in vivo- and in vitro-derived isolates of EIAV. Results of these studies may reveal if cellular transcription factors play a role in regulation of viral gene expression. If so, gel retardation assays and DNA footprinting will be used to confirm the presence of cellular DNA binding proteins and to identify LTR sequences necessary for cell-specific regulation of viral expression. Variability in the S3 open reading frame of EIAV suggests that both rev-competent and rev-defective genotypes co-exist in vivo. To determine if this is true, the applicant proposes to subclone S3 variants into a full-length proviral clone of EIAV. Rev function will be analyzed by quantitating differentially spliced mRNA transcripts in nuclear and cytoplasmic fractions of transfected cells. The effect of rev variation on expression of viral proteins will be determined using radioimmunoprecipitation. The investigator anticipates that these studies may indicate that rev-defective genotypes play an important role in vivo in restriction of viral gene expression and maintenance of persistent infection.
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