Complex Expression of the UL136 Gene of Human Cytomegalovirus Results in Multiple Protein Isoforms with Unique Roles in Replication

Abstract

Human cytomegalovirus (HCMV) is a complex DNA virus with a 230-kb genome encoding 170 and up to 750 proteins. The upper limit of this coding capacity suggests the evolution of complex mechanisms to substantially increase the coding potential from the 230-kb genome. Our work examines the complexity of one gene, UL136, encoded within the ULb= region of the genome that is lost during serial passage of HCMV in cultured fibroblasts. UL136 is expressed as five protein isoforms. We mapped these isoforms and demonstrate that they originate from both a complex transcriptional profile and, possibly, the usage of multiple translation initiation sites. Intriguingly, the pUL136 isoforms exhibited distinct subcellular distributions with varying association with the Golgi apparatus. The subcellular localization of membrane-bound isoforms of UL136 differed between when they were expressed exogenously and when they were expressed in the context of viral infection, suggesting that the trafficking of these isoforms is mediated by infection-specific factors. While UL136, like most ULb= genes, was dispensable for replication in fibroblasts, the soluble 23-and 19-kDa isoforms suppressed virus replication. In CD34؉ hematopoietic progenitor cells (HPCs) infected in vitro, disruption of the 23-and 19-kDa isoforms resulted in increased replication and a loss of the latency phenotype, similar to the effects of the UL138 latency determinant encoded within the same genetic locus. Our work suggests a complex interplay between the UL136 isoforms which balances viral replication in multiple cell types and likely contributes to the cell typedependent phenotypes of the UL133/8 locus and the outcome of HCMV infection. IMPORTANCEHCMV is a significant cause of morbidity in immunocompromised individuals, including transplant patients. The lifelong persistence of the virus results in a high seroprevalence worldwide and may contribute to age-related pathologies, such as atherosclerosis. The mechanisms of viral persistence are poorly understood; however, understanding the molecular basis of persistence is imperative for the development of new treatments. In this work, we characterize a complex HCMV gene, UL136, which is expressed as five protein isoforms. These isoforms arise predominantly from complex transcriptional mechanisms, which contribute to an increased coding capacity of the virus. Further, the UL136 isoforms oppose the activity of one another to balance HCMV replication in multiple cell types. We identify soluble isoforms of UL136 that function to suppress virus replication in fibroblasts and in CD34 ؉ HPCs for latency.

Long and Short Isoforms of the Human Cytomegalovirus UL138 Protein Silence IE Transcription and Promote Latency

“…To create AD169-UL138HA-M16I, methionine 16 was mutated to an isoleucine using the following primers: 5=-ACG ATC TGC CGC TGA ACG TCG GGT TAC CCA TCA TCG GCG TGA TCC TCG TGC TGA TCG TGG CCA TTC TAG GGA TAA CAG GGT AAT CGA TTT-3= and 5=-CAA TGG TAA GCT AGA TAG CAG AGA ATG GCC ACG ATC AGC ACG AGG ATC ACG CCG ATG ATG GGT AA GCC AGT GTT ACA ACC AAT TAA CC-3=. A derivative of TB40/E expressing green fluorescent protein (GFP) has been previously described (40) and was used to generate additional mutants using a two-step galK-based approach (9). Primers utilized are listed in Table 1.…”

“…Normal human dermal fibroblasts (NHDFs) (Clonetics), human fetal lung fibroblasts (MRC-5) (CCL-171; ATCC), THP-1 monocytes (TIB-202; ATCC), and human embryonic stem cells (ESCs) (WA01; WiCell) were maintained as previously described (9,20,31). Primary human CD34 ϩ cells derived from cord blood were either purchased from Lonza (2C-101) and cultured as previously described (21) or isolated from deidentified donors at the University of Arizona Medical Center and cultured as previously described (38).…”

“…Cells were lysed in either radioimmunoprecipitation assay (RIPA; Thermo), 1% SDS, or 1ϫ passive lysis (Promega) buffer, and equal amounts of lysates were separated by SDS-PAGE, transferred to Optitran (GE Healthcare) or polyvinylidene difluoride (PVDF; Millipore) membranes, and analyzed by Western blotting as previously described (9,30). For indirect immunofluorescence, adherent NHDFs and suspension THP-1 or CD34 ϩ cells were washed with phosphate-buffered saline (PBS), fixed in 1% paraformaldehyde, and processed as previously described (20,21,29).…”

“…Genomic approaches have also cataloged previously undetected transcribed and translated sequences during both lytic and latent infections (34)(35)(36)(37). The UL136 gene described above generates a series of coterminal protein isoforms with both overlapping and distinct functions (9,15), illustrating the functional impact of previously unrecognized viral coding capacity. Here we show that UL138 gene also produces two coterminal proteins originating from separate in-frame methionine codons that support HCMV latency with different temporal phenotypes.…”

“…While many of the mechanisms important to productive infection are well understood in fibroblasts (3), the mechanisms critical to latency remain poorly defined. However, in the last decade, an in vitro system to model experimental HCMV latency emerged, permitting genetic (9)(10)(11)(12)(13)(14)(15) and molecular (16)(17)(18) analyses of latently infected cells. During latency, the expression of productive-phase genes is suppressed.…”