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

Wang, Depeng; Li, Gang; Schauflinger, Martin; Nguyen, Christopher; Hall, Ellie D.; Yurochko, Andrew D.; Einem, Jens von; Kamil, Jeremy P.

doi:10.1128/jvi.03477-12

Cited by 25 publications

(50 citation statements)

References 72 publications

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“…UL97 mutants exhibit a severe growth defect (53), which is greater in clinical strains than in laboratory-adapted strains of HCMV (54). As demonstrated by our collaborative work in the accompanying article (52), UL97 promotes the second phase of IE2 expression, viral DNA synthesis, and progression of the lytic infection cycle.…”

Section: Discussionmentioning

confidence: 95%

Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

Umashankar

Rak

Bughio

et al. 2014

J Virol

139

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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.

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Section: Discussionmentioning

confidence: 95%

Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

Umashankar

Rak

Bughio

et al. 2014

J Virol

139

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“…A BAC clone of TB40/E, TB40-BAC4 (16), was a gift of Christian Sinzger (University of Ulm, Ulm, Germany), and its UL97-null (⌬97) mutant derivative has been previously described (17). TB40/E with a deletion of the entire UL133/8 locus (TB40/E_UL133-UL138 NULL [UL133/ 8 NULL ]), a TB40/E mutant unable to express UL138 (TB40/E_UL138 STOP [UL138 STOP ]), and a TB40/E mutant that does not express either UL135 or UL138 (TB40/E_UL135 STOP /UL138 STOP [5/8 STOP ]) are described elsewhere (18,19).…”

Section: Cells and Virusmentioning

confidence: 99%

“…The BAC was also analyzed by restriction enzyme digestion to confirm that spurious changes had not occurred and was reconstituted to generate infectious virus, as described previously (17). Viral stocks were prepared by ultracentrifuge concentration, and titers were determined by assays that measured the 50% tissue culture infective dose (TCID 50 ) or the number of infectious units (IU)/ml, which have been described elsewhere (17). Replication kinetics experiments were performed as previously described (17).…”

Section: Cells and Virusmentioning

confidence: 99%

“…The verified sequence spanned from nucleotide positions 42388 to 42882 of the AD169 genome (the numbering is according to that for the sequence with GenBank accession number AC146999.1 and does not reflect the insertion of UL133/8). The BAC was also analyzed by restriction enzyme digestion to confirm that spurious changes had not occurred and was reconstituted to generate infectious virus, as described previously (17). Viral stocks were prepared by ultracentrifuge concentration, and titers were determined by assays that measured the 50% tissue culture infective dose (TCID 50 ) or the number of infectious units (IU)/ml, which have been described elsewhere (17).…”

Section: Cells and Virusmentioning

confidence: 99%

“…TB40/E with a deletion of the entire UL133/8 locus (TB40/E_UL133-UL138 NULL [UL133/ 8 NULL ]), a TB40/E mutant unable to express UL138 (TB40/E_UL138 STOP [UL138 STOP ]), and a TB40/E mutant that does not express either UL135 or UL138 (TB40/E_UL135 STOP /UL138 STOP [5/8 STOP ]) are described elsewhere (18,19). AD169_UL133/8 was constructed using en passant Red mutagenesis in Escherichia coli (20,21), as previously described (17). Briefly, a PCR product containing an I-SceI-aphAI cassette was generated using primers TB_UL135Kan_Fw (5=-GCT CTA CAT AGG AGA GGA TGG TCT GCC GAT AGA TAA ACC CGA GTT TCC TCC GGC GCG ATT TAG GGA TAA CAG GGT AAT CGA TTT-3=) and TB_ UL135Kan_Rv (5=-TG GAT ACG TCG GGG ATC TCG AAT CGC GCC GGA GGA AAC TCG GGT TTA TCT ATC GGC AGA CGC CAG TGT TAC AAC CAA TTA ACC-3=) and inserted via Red recombination into the UL135 coding region of TB40/E.…”

Section: Cells and Virusmentioning

confidence: 99%

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An Epistatic Relationship between the Viral Protein Kinase UL97 and the UL133-UL138 Latency Locus during the Human Cytomegalovirus Lytic Cycle

Rak

Nguyen

et al. 2014

J Virol

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We report that UL133-UL138 (UL133/8), a transcriptional unit within the ULb= region (ULb=) of the human cytomegalovirus (HCMV) genome, and UL97, a viral protein kinase encoded by HCMV, play epistatic roles in facilitating progression of the viral lytic cycle. In studies with HCMV strain TB40/E, pharmacological blockade or genetic ablation of UL97 significantly reduced the levels of mRNA and protein for IE2 and viral early and early-late genes during a second wave of viral gene expression that commenced at between 24 and 48 h postinfection. These effects were accompanied by significant defects in viral DNA synthesis and viral replication. Interestingly, deletion of UL133/8 likewise caused significant defects in viral DNA synthesis, viral gene expression, and viral replication, which were not exacerbated upon UL97 inhibition. When UL133/8 was restored to HCMV laboratory strain AD169, which otherwise lacks the locus, the resulting recombinant virus replicated similarly to the parental virus. However, during UL97 inhibitor treatment, the virus in which UL133/8 was restored showed significantly exacerbated defects in viral DNA synthesis, viral gene expression, and production of infectious progeny virus, thus recapitulating the differences between wild-type TB40/E and its UL133/8-null derivative. Phenotypic evaluation of mutants null for specific open reading frames within UL133/8 revealed a role for UL135 in promoting viral gene expression, viral DNA synthesis, and viral replication, which depended on UL97. Taken together, our findings suggest that UL97 and UL135 play interdependent roles in promoting the progression of a second phase of the viral lytic cycle and that these roles are crucial for efficient viral replication. IMPORTANCEA unique feature of the herpesviruses, such as human cytomegalovirus (HCMV), is that they can undergo latency, a state during which the virus silences its gene expression, which allows lifelong viral persistence in immunocompetent hosts. We have uncovered an unexpected link between a cluster of HCMV genes involved in latency, UL133-UL138, and a virally encoded protein kinase, UL97, which plays crucial roles in manipulating the cell cycle during HCMV lytic replication. Although viral immediate early (IE) gene expression is essential for HCMV lytic replication, the activation of IE gene expression in latently infected cells is not sufficient to result in production of infectious virus. Our findings here and in an accompanying study (M. Umashankar, M. Rak, F. Bughio, P. Zagallo, K. Caviness, and F. D. Goodrum, J. Virol. 88:5987-6002, 2014) show that proteins expressed from the UL133-UL138 latency locus and UL97 play interdependent roles in overcoming checkpoints that restrict the viral lytic replication cycle, findings which suggest intriguing implications for establishment of and reactivation from HCMV latency.

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Serine 13 of the human cytomegalovirus viral cyclin-dependent kinase UL97 is required for regulatory protein 14-3-3 binding and UL97 stability

Iwahori

Umaña

Kalejta

et al. 2022

Journal of Biological Chemistry

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

Cited by 25 publications

References 72 publications

Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

Antagonistic Determinants Controlling Replicative and Latent States of Human Cytomegalovirus Infection

An Epistatic Relationship between the Viral Protein Kinase UL97 and the UL133-UL138 Latency Locus during the Human Cytomegalovirus Lytic Cycle

Serine 13 of the human cytomegalovirus viral cyclin-dependent kinase UL97 is required for regulatory protein 14-3-3 binding and UL97 stability

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