The Sequence and Antiapoptotic Functional Domains of the Human Cytomegalovirus UL37 Exon 1 Immediate Early Protein Are Conserved in Multiple Primary Strains

Abstract

The human cytomegalovirus UL37 exon 1 gene encodes the immediate early protein pUL37x1 that has antiapoptotic and regulatory activities. Deletion mutagenesis analysis of the open reading frame of UL37x1 identified two domains that are necessary and sufficient for its antiapoptotic activity. These domains are confined within the segments between amino acids 5 to 34, and 118 to 147, respectively. The first domain provides the targeting of the protein to mitochondria. Direct PCR sequencing of UL37 exon 1 amplified from 26 primary strains of human cytomegalovirus demonstrated that the promoter, polyadenylation signal, and the two segments of pUL37x1 required for its antiapoptotic function were invariant in all sequenced strains and identical to those in AD169 pUL37x1. In total, UL37 exon 1 varies between 0.0 and 1.6% at the nucleotide level from strain AD169. Only 11 amino acids were found to vary in one or more viral strains, and these variations occurred only in the domains of pUL37x1 dispensable for its antiapoptotic function. We infer from this remarkable conservation of pUL37x1 in primary strains that this protein and, probably, its antiapoptotic function are required for productive replication of human cytomegalovirus in humans.

Mitochondrial and Secretory Human Cytomegalovirus UL37 Proteins Traffic into Mitochondrion-Associated Membranes of Human Cells

“…The epitope for Ab1064 and DC35 (aa 27 to 40) and the C-terminal Flag tag (flag) are shown. For the pUL37x1 mutants, the sequences in pUL37x1 ⌬2-23 and pUL37x1 ⌬23-34 (19,25), as well as the C-terminal Myc tag (hexagon), are shown. a final 10-min extension at 72°C.…”

“…To determine the UL37x1 targeting signal for the MAM, we examined the trafficking of two C-terminally tagged pUL37x1 deletion mutants, known to be partially defective in mitochondrial importation (19). PSS-1 20 cells were transfected with a vector expressing pUL37x1 ⌬2-23-myc and fractionated.…”

“…Consistent with this membrane anchoring is the finding that wild-type, untagged pUL37x1 is not detectable in the cytosol fraction of either transfected or HCMV-infected cells. However, two Myc-tagged pUL37x1 deletion mutants, defective in their trafficking to MAM and mitochondria, were aberrantly detected in the cytosol (19,25).…”

“…These sequences include a strongly hydrophobic leader (aa 1 to 22) and a juxtaposed basic domain (aa 23 to 34), which jointly serve as a bipartite signal to target UL37 proteins to the ER and mitochondria (25,27). This NH 2 -terminal targeting domain constitutes the first UL37x1 antiapoptotic domain, which when combined with a downstream domain (aa 118 to 147) are sufficient to confer its antiapoptotic activity (5,18,19,28,32,34,45). Between the UL37x1 antiapoptotic domains lies an acidic domain (aa 81 to 108), which plays a role in the transactivation of HCMV early gene promoters, whose products are needed for its oriLyt replication (13,31,52).…”

“…Between the UL37x1 antiapoptotic domains lies an acidic domain (aa 81 to 108), which plays a role in the transactivation of HCMV early gene promoters, whose products are needed for its oriLyt replication (13,31,52). pUL37x1, gpUL37, and pUL37 M dually traffic to the ER and to the mitochondrial outer membrane in transfected and in HCMV-infected cells (2,18,19,(25)(26)(27)42). Most mitochondrial proteins, including soluble and membrane-bound proteins, are synthesized in the cytosol, transported to mitochondria by heat shock proteins, and imported via translocation complexes in the outer and inner mitochondrial membranes (30,36).…”