Complex Interplay of the UL136 Isoforms Balances Cytomegalovirus Replication and Latency

Abstract

Human cytomegalovirus (HCMV), a betaherpesvirus, persists indefinitely in the human host through poorly understood mechanisms. The UL136 gene is carried within a genetic locus important to HCMV latency termed the UL133/8 locus, which also carries UL133, UL135, and UL138. Previously, we demonstrated that UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and, likely, translation initiation mechanisms. We previously showed that the UL136 isoforms are largely dispensable for virus infection in fibroblasts, a model for productive virus replication. In our current work, UL136 has emerged as a complex regulator of HCMV infection in multiple contexts of infection relevant to HCMV persistence: in an endothelial cell (EC) model of chronic infection, in a CD34+ hematopoietic progenitor cell (HPC) model of latency, and in an in vivo NOD-scid IL2Rγcnull humanized (huNSG) mouse model for latency. The 33- and 26-kDa isoforms promote replication, while the 23- and 19-kDa isoforms suppress replication in ECs, in CD34+ HPCs, and in huNSG mice. The role of the 25-kDa isoform is context dependent and influences the activity of the other isoforms. These isoforms localize throughout the secretory pathway, and loss of the 33- and 26-kDa UL136 isoforms results in virus maturation defects in ECs. This work reveals an intriguing functional interplay between protein isoforms that impacts virus replication, latency, and dissemination, contributing to the overall role of the UL133/8 locus in HCMV infection.

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

“…Our current study demonstrates that the two protein isoforms of pUL138 may have both overlapping and distinct functions during HCMV infection. In recent years, many studies have contributed to a body of work that begins to effectively demonstrate the power and function of increased coding capacity in HCMV through transcriptional, translational, and posttranslational mechanisms (9,15,32,33,37,58,59). Further, many examples of viral protein isoforms that have similar or unique functions exist (60)(61)(62)(63)(64)(65)(66)(67), but these examples likely only begin to scratch the surface of the important roles that protein isoforms play in viral infection and virus-host interactions.…”

“…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.…”

“…Genetic analyses indicate that UL135 and some UL136 isoforms are required for efficient productive infection in endothelial cells (23)(24)(25) and that UL138, when expressed in the presence of UL133 and UL136 but in the absence of UL135, impairs efficient productive infection of fibroblasts (13). Notably, viruses deficient in UL133, UL138, or the soluble 23-/19-kDa isoforms of UL136 generate many infectious virus progeny in CD34 ϩ hematopoietic cells, whereas wild-type viruses generate few such progeny (11,12,15).…”

“…Genetic analyses indicate that UL135 and some UL136 isoforms are required for efficient productive infection in endothelial cells (23)(24)(25) and that UL138, when expressed in the presence of UL133 and UL136 but in the absence of UL135, impairs efficient productive infection of fibroblasts (13). Notably, viruses deficient in UL133, UL138, or the soluble 23-/19-kDa isoforms of UL136 generate many infectious virus progeny in CD34 ϩ hematopoietic cells, whereas wild-type viruses generate few such progeny (11,12,15). Molecular studies have revealed that UL135 and UL136 promote cytoplasmic maturation of viral particles in endothelial cells (23,24), UL135 impairs immune synapse formation (26), and UL138 promotes cell surface expression of the tumor necrosis factor alpha (TNF-␣) receptor (TNFR) (27,28) as well as impairs cell surface expression of the multidrug resistance-associated protein-1 (MRP1) (18).…”

“…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.…”