Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

Abstract

The mechanisms by which viruses persist and particularly those by which viruses actively contribute to their own latency have been elusive. Here we report the existence of opposing functions encoded by genes within a polycistronic locus of the human cytomegalovirus (HCMV) genome that regulate cell type-dependent viral fates: replication and latency. The locus, referred to as the UL133-UL138 (UL133/8) locus, encodes four proteins, pUL133, pUL135, pUL136, and pUL138. As part of the ULb= region of the genome, the UL133/8 locus is lost upon serial passage of clinical strains of HCMV in cultured fibroblasts and is therefore considered dispensable for replication in this context. Strikingly, we could not reconstitute infection in permissive fibroblasts from bacterial artificial chromosome clones of the HCMV genome where UL135 alone was disrupted. The loss of UL135 resulted in complex phenotypes and could ultimately be overcome by infection at high multiplicities. The requirement for UL135 but not the entire locus led us to hypothesize that another gene in this locus suppressed virus replication in the absence of UL135. The defect associated with the loss of UL135 was largely rescued by the additional disruption of the UL138 latency determinant, indicating a requirement for UL135 for virus replication when UL138 is expressed. In the CD34 ؉ hematopoietic progenitor model of latency, viruses lacking only UL135 were defective for viral genome amplification and reactivation. Taken together, these data indicate that UL135 and UL138 comprise a molecular switch whereby UL135 is required to overcome UL138-mediated suppression of virus replication to balance states of latency and reactivation. IMPORTANCEMechanisms by which viruses persist in their host remain one of the most poorly understood phenomena in virology. Herpesviruses, including HCMV, persist in an incurable, latent state that has profound implications for immunocompromised individuals, including transplant patients. Further, the latent coexistence of HCMV may increase the risk of age-related pathologies, including vascular disease. The key to controlling or eradicating HCMV lies in understanding the molecular basis for latency. In this work, we describe the complex interplay between two viral proteins, pUL135 and pUL138, which antagonize one another in infection to promote viral replication or latency, respectively. We previously described the role of pUL138 in suppressing virus replication for latency. Here we demonstrate a role of pUL135 in overcoming pUL138-mediated suppression for viral reactivation. From this work, we propose that pUL135 and pUL138 constitute a molecular switch balancing states of latency and reactivation.

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

“…While UL138 has been associated with multiple effects during HCMV infection (13,16,18), previous studies did not take into account the individual roles of the pUL138 protein isoforms. Our current study demonstrates that the two protein isoforms of pUL138 may have both overlapping and distinct functions during HCMV infection.…”

“…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).…”

“…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.…”

Recent advances in CMV tropism, latency, and diagnosis during aging

“…The Goodrum lab is focused on identifying viral determinants of latency-virus genes that promote viral latency or reactivation from latency-as a means to understand the basic molecular mechanisms contributing to latency (Goodrum et al 2007;Umashankar et al 2011;Umashankar et al 2014). HCMV coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication (Umashankar et al 2014).…”

“…HCMV coordinates the expression of two viral genes, UL135 and UL138, which have opposing roles in regulating viral replication (Umashankar et al 2014). UL135 promotes reactivation from latency and virus replication, in part, by overcoming replication-suppressive effects of UL138.…”