Positive and Negative Regulation of Chicken Anemia Virus Transcription

Abstract

Chicken anemia virus (CAV) is a small circular single-stranded DNA virus with a single promoter-enhancer region containing four consensus cyclic AMP response element sequences (AGCTCA), which are similar to the estrogen response element (ERE) consensus half-sites (A)GGTCA. These sequences are arranged as direct repeats, an arrangement that can be recognized by members of the nuclear receptor superfamily. Transienttransfection assays which use a short CAV promoter construct that ended at the transcription start site and drive expression of enhanced green fluorescent protein (EGFP) showed high basal activity in DF-1, LMH, LMH/2A, and primary theca and granulosa cells. The estrogen receptor-enhanced cell line, LMH/2A, had significantly greater expression than LMH cells, and this expression was significantly increased with estrogen treatment. A long promoter construct which included GGTCA-like sequences downstream of the first CAV protein translation start site was found to have significantly less EGFP expression in DF-1 cells than the short promoter, which was largely due to decreased RNA transcription. DNA-protein binding assays indicated that proteins recognizing a consensus ERE palindrome also bind GGTCA-like sequences in the CAV promoter. Estrogen receptor and other members of the nuclear receptor superfamily may provide a mechanism to regulate CAV activity in situations of low virus copy number.

Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding δEF1

“…This suggested that members of the nuclear receptor superfamily provide a mechanism to regulate CAV activity when low viral genome copy numbers are present (Miller et al, 2005). In addition, comparison of expression from a short (pEGFP-SE) and long (pEGFP-LE) CAV promoter construct, found less expression from pEGFP-LE that was largely due to decreased mRNA transcription (Miller et al, 2005).…”

“…This suggested that members of the nuclear receptor superfamily provide a mechanism to regulate CAV activity when low viral genome copy numbers are present (Miller et al, 2005). In addition, comparison of expression from a short (pEGFP-SE) and long (pEGFP-LE) CAV promoter construct, found less expression from pEGFP-LE that was largely due to decreased mRNA transcription (Miller et al, 2005). Sequences at the transcription start point (TSP) present in pEGFP-LE were found to have a potential E boxlike element similar to site-binding d-crystalline enhancer binding protein (dEF1) To examine whether TR and COUP-TF1 can modulate CAV promoter expression, we used co-transfection experiments of CAV promoter constructs with expression vectors for TR and COUP-TF1.…”

“…The most wellcharacterized is chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) (Cooney et al, 1993;Park et al, 2003). Together, these nuclear receptors can bind a wide range of ERE-like motifs and often compete for binding to the same DNA sequence.Due to the common occurrence of seroconversion at the time of sexual maturation in specific-pathogen-free flocks (Miller et al, 2001) and the presence of viral DNA in male and female gonads (Cardona et al, 2000b) and in freshly laid fertilized eggs (Miller et al, 2003), we hypothesized that CAV gene regulation is influenced by the regulation of Miller et al, 2005), and that the ERE-like sequences from the CAV promoter could compete for binding to unidentified proteins recognizing a consensus ERE. This suggested that members of the nuclear receptor superfamily provide a mechanism to regulate CAV activity when low viral genome copy numbers are present (Miller et al, 2005).…”

“…All constructs were verified by sequencing (Biotechnology Resource Center at Cornell University). The plasmids pEGFP-N1 (BD Biosciences), with EGFP expression under control of the immediate-early cytomegalovirus (CMV) promoter, and the promoterless pEGFP-1 were used as the positive and negative controls, respectively.Transactivation assays were performed as described previously (Miller et al, 2005). Briefly, cells were transfected using Lipofectamine Plus or Lipofectamine 2000 (Invitrogen) and 0.3 mg EGFP reporter plasmid, with 0.2 mg pRS-COUP-TF1, pEX-cTRa or the negative control pRc-RSV in each well of six-well plates.…”

“…Two separate clones were tested for each vector and an empty vector containing the Rous sarcoma virus (RSV) promoter pRc-RSV (Invitrogen) was used to co-transfect negative controls. Construction of the reporter plasmids containing the CAV short and long promoters, pEGFP-SE and pEGFP-LE, has been previously described by Miller et al (2005). Deletion mutants were designed with 63 or 71 nucleotide (nt) deletions from the 39 end of the long promoter and identified as pEGFP-LE-63 and pEGFP-LE-71, respectively (Fig.…”