Human Cytomegalovirus UL97 Kinase and Nonkinase Functions Mediate Viral Cytoplasmic Secondary Envelopment

139

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.

Section: Discussionmentioning

confidence: 99%

Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

Umashankar

Rak

Bughio

et al. 2014

139

“…Although UL97 has been implicated in several other steps during the virus replication cycle (10)(11)(12)(13)(14)(15)(16), in dividing cells the magnitude of the nuclear egress defect due to a UL97 null mutation or a UL97 inhibitor is very similar to the magnitude of the virus yield defect (10,17). In serum-starved, nondividing cells, part of the virus yield defect can be ascribed to a defect in viral DNA synthesis, stemming from UL97's role in inactivation of retinoblastoma protein (16)(17)(18).…”

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confidence: 99%

Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

Sharma

Bender

Kamil

et al. 2015

Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC. IMPORTANCEHuman cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses. Herpesviruses replicate and package their DNA genomes into capsids in the nucleus of the host cell. The nucleocapsids are then transported out of the nucleus through an unusual process called nuclear egress. A widely accepted model for nuclear egress entails envelopment and de-envelopment of capsids as they transit the nuclear membranes (reviewed in references 1, 2, and 3). Studies of human cytomegalovirus (HCMV) nuclear egress are of particular interest, because of the medical ...

“…The current understanding of the biological roles of UL97 comes from studies of infections in cultured cells using UL97-null deletion mutants (⌬97), kinase-deficient point mutants, or wildtype (wt) viruses in the presence of UL97 inhibitors (5,(7)(8)(9)(18)(19)(20)(21)(22)(23)(24)(25)(26). However, these studies have been conducted almost exclusively with extensively passaged, "laboratory strains" of HCMV, such as AD169 and Towne.…”

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confidence: 99%

“…However, these studies have been conducted almost exclusively with extensively passaged, "laboratory strains" of HCMV, such as AD169 and Towne. UL97-deficient infections with such strains exhibit defects in myriad viral processes during replication, including nuclear egress, viral DNA synthesis, virion morphogenesis, and cytoplasmic egress of enveloped virions, such that UL97 has been hypothesized to play roles in each of these processes (7,9,(18)(19)(20)(21)(22)(23)27). Identification of the retinoblastoma tumor suppressor protein (pRb) and nuclear A-type lamins as cellular substrates of UL97 kinase activity has suggested mechanisms by which UL97 might contribute to viral DNA synthesis and nuclear egress, respectively (21,23,27,28).…”

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confidence: 99%

The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

Wang

Schauflinger³

et al. 2013

bWe report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb= region of the viral genome. A UL97-null (⌬97) mutant of strain TB40/E, which encodes a full-length ULb= region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a ⌬97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb= region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(␤-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb= regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ϳ100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb= regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb= region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb= region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis.