The E3 CR1-gamma gene in human adenoviruses associated with epidemic keratoconjunctivitis

Abstract

Human adenovirus species D type 37 (HAdV-D37) is an important etiologic agent of epidemic keratoconjunctivitis. Annotation of the whole genome revealed an open reading frame (ORF) in the E3 transcription unit predicted to encode a 31.6 kDa protein. This ORF, also known as CR1-γ, is predicted to be an integral membrane protein containing N-terminal signal sequence, luminal, transmembrane, and cytoplasmic domains. HAdV-D19 (C), another viral pathogen causing epidemic keratoconjunctivitis, contains an ORF 100% identical to its HAdV-D37 homologue but only 66% identical to other HAdV-D homologues. Kinetics of RNA expression and confirmation of splicing to the adenovirus tripartite leader sequence suggest a role for the protein product of CR1-γ in the late stages of the viral replication cycle. Confocal microscopy is consistent with expression in the cytoplasm. Sequence analysis reveals a hypervariable luminal domain and a conserved cytoplasmic domain. The luminal domain is predicted to contain multiple N-glycosylation sites. The cytoplasmic domain contains a putative protein kinase C phosphorylation site and potential YXXϕ and dileucine (LL) motifs suggesting a potential role in modification of host proteins.

Introduction

The E3 transcription unit of human adenoviruses (HAdVs) encodes several proteins that have been shown to modulate the host immune response. Three open reading frames (ORFs), RID-α, RID-β, and 14.7K, are conserved across every species of HAdV. Gene products of these ORFs block apoptosis by down-regulation of FAS, TRAIL 1, and TRAIL 2 receptors and blockade of TNF-α (Benedict et al., 2001, Hilgendorf et al., 2003). Another ORF within the E3 region encodes glycoprotein (gp)19K, known to block presentation of MHC class I molecules on the cell surface (Burgert et al., 1987, Deryckere and Burgert, 1996, Feuerbach et al., 1994), and has homologues in HAdV-B, -C, -D, and -E. While labeled as an “early” transcription region, transcripts from the E3 family are expressed both early and late during viral infection (Bhat and Wold, 1986, Chow and Broker, 1978, Chow et al., 1977, Chow et al., 1979, Chow et al., 1980). However, proteins encoded in this region do not appear to be required for viral replication (Wold and Gooding, 1991).

While several proteins within the adenovirus genome have been extensively studied, many of the genes located within the E3 region remain poorly characterized. The role of those few E3 proteins with known function were determined mainly in HAdV-C. The E3 transcription unit, though, represents an area of major divergence between HAdV-D and other HAdV species (Burgert and Blusch, 2000, Robinson et al., 2008, Robinson et al., 2009b). The number of potential genes within the E3 region also differs among HAdV species. For HAdV-Ds, E3 is predicted to encode 8 ORFs encompassing approximately 5000 bps. Four of those genes have no known function.

HAdV-D19 and HAdV-D37, along with types D8, D53, D54, and D56 are all etiological agents of epidemic keratoconjunctivitis (EKC), a common and highly communicable eye infection. HAdV-D19 and HAdV-D37 are also isolated from the genitourinary tract (de Jong et al., 1981, Phillips et al., 1982, Swenson et al., 1995). Recently our lab has sequenced the complete genome of HAdV-D37, and of a clinical isolate of HAdV-D19 (strain C) causing EKC, termed HAdV-D19 (C) (Robinson et al., 2008, Robinson et al., 2009b). During annotation of these genomes we identified a putative ORF located between the CR1-β (also known as 49K) and RID-α predicted to encode a protein of 31.6 kDa in size. This gene has been previously identified as 31.6K or CR1-γ (Burgert and Blusch, 2000, Davison et al., 2003). Here we describe analysis of the CR1-γ gene from HAdV-D37 and contrast it with homologues from other HAdV-D genomes. Analysis of RNA expression kinetics reveals that the gene is expressed from the tripartite leader sequence, suggesting a function in the late stages of the viral replication cycle. In silico analysis suggests an integral membrane protein that is highly glycosylated, similar to other E3 proteins. We also show diversity between homologues of the gene within HAdV-D, suggesting that immune pressure may have driven the evolution of this gene.