Rapid detection and quantitation of ganciclovir resistance in cytomegalovirus quasispecies

Ruiz-Carrascoso, Guillermo; Romero-Gómez, María Pilar; Plaza, Diego; Mingorance, Jesús

doi:10.1002/jmv.23570

Cited by 9 publications

(8 citation statements)

References 37 publications

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“…This region overlaps with a minimal binding region for cyclin T1. Recent modeling approaches based on the in silico prediction of binding interfaces suggested extended binding interfaces for cyclins T1, B1 and H. Moreover, pUL97 is involved in the multiple regulatory steps during HCMV replication through the phosphorylation of viral and cellular substrates (see horizontal bars), as reported by several independent groups [44][45][46]54,55,57,75,[79][80][81][82][83][84][94][95][96][97][98][99][100][101]122,[126][127][128][129][130][131][132]. Substrates include the viral DNA polymerase cofactor pUL44, viral RNA transport factor pUL69, major tegument protein pp65, nuclear egress core protein heterodimer pUL50-pUL53, cellular multi-ligand binding protein p32/gC1qR, tumor suppressor/checkpoint protein Rb, nuclear lamins A/C, RNA polymerase II, translation factor EF-1δ, interferon-inducible proteins IFI16 and SAMHD1, as well as the therapeutically applied nucleoside analog ganciclovir (GCV; [47,56,82] and references therein).…”

Section: Involvement In Intrinsic Immunity Evasionmentioning

confidence: 72%

The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting

Steingruber

Marschall

2020

Microorganisms

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Human cytomegalovirus (HCMV) expresses a variety of viral regulatory proteins that undergo close interaction with host factors including viral-cellular multiprotein complexes. The HCMV protein kinase pUL97 represents a viral cyclin-dependent kinase ortholog (vCDK) that determines the efficiency of HCMV replication via phosphorylation of viral and cellular substrates. A hierarchy of functional importance of individual pUL97-mediated phosphorylation events has been discussed; however, the most pronounced pUL97-dependent phenotype could be assigned to viral nuclear egress, as illustrated by deletion of the UL97 gene or pharmacological pUL97 inhibition. Despite earlier data pointing to a cyclin-independent functionality, experimental evidence increasingly emphasized the role of pUL97-cyclin complexes. Consequently, the knowledge about pUL97 involvement in host interaction, viral nuclear egress and additional replicative steps led to the postulation of pUL97 as an antiviral target. Indeed, validation experiments in vitro and in vivo confirmed the sustainability of this approach. Consequently, current investigations of pUL97 in antiviral treatment go beyond the known pUL97-mediated ganciclovir prodrug activation and henceforward include pUL97-specific kinase inhibitors. Among a number of interesting small molecules analyzed in experimental and preclinical stages, maribavir is presently investigated in clinical studies and, in the near future, might represent a first kinase inhibitor applied in the field of antiviral therapy.

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Section: Involvement In Intrinsic Immunity Evasionmentioning

confidence: 72%

The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting

Steingruber

Marschall

2020

Microorganisms

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“…Although it was difficult to detect a small population of variants in the host genome using a traditional approach such as PCR, NGS, owing to its depth, enabled us to detect a novel genomic variation (27). NGS has strongly supported the studies of viral genetic diversity, especially in RNA viruses (28,29), whereas the detection by NGS of quasispecies in DNA virus has been reported less frequently (30)(31)(32)(33). Using PCR analysis, the presence of VP1 quasispecies has been reported in polyomavirus BK (BKV) (34).…”

Section: Discussionmentioning

confidence: 99%

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

Takahashi

Sekizuka

Fukumoto

et al. 2017

J Virol

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JC virus (JCV) is a DNA virus causing progressive multifocal leukoencephalopathy (PML) in immunodeficient patients. In the present study, 22 genetic quasispecies with more than 1.5% variant frequency were detected in JCV genomes from six clinical samples of PML by next-generation sequencing. A mutation from A to C at nucleotide (nt) 3495 in JCV Mad1 resulting in a V-to-G amino acid substitution at amino acid (aa) position 392 of the large T antigen (TAg) was identified in all six cases of PML at 3% to 19% variant frequencies. Transfection of JCV Mad1 DNA possessing the V392G substitution in TAg into IMR-32 and human embryonic kidney 293 (HEK293) cells resulted in dramatically decreased production of JCV-encoded proteins. The virus DNA copy number was also reduced in supernatants of the mutant virus-transfected cells. Transfection of the IMR-32 and HEK293 cells with a virus genome containing a revertant mutation recovered viral production and protein expression. Cotransfection with equal amounts of wild-type genome and mutated JCV genome did not reduce the expression of viral proteins or viral replication, suggesting that the mutation did not have any dominantnegative function. Finally, immunohistochemistry demonstrated that TAg was expressed in all six pathological samples in which the quasispecies were detected. In conclusion, the V392G amino acid substitution in TAg identified frequently in PML lesions has a function in suppressing JCV replication, but the frequency of the mutation was restricted and its role in PML lesions was limited.IMPORTANCE DNA viruses generally have lower mutation frequency than RNA viruses, and the detection of quasispecies in JCV has rarely been reported. In the present study, a next-generation sequencer identified a JCV quasispecies with an amino acid substitution in the T antigen in patients with PML. In vitro studies showed that the mutation strongly repressed the expression of JC viral proteins and reduced the viral replication. However, because the frequency of the mutation was low in each case, the total expression of virus proteins was sustained in vivo. Thus, JC virus replicates in PML lesions in the presence of a mutant virus which is able to repress virus replication.

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“…Moreover, pUL97 is involved in the multiple regulatory steps during HCMV replication, as exerted through the phosphorylation of viral and cellular substrates (see horizontal bars for those binding regions within pUL97 that could be mapped so far), i.e. including the viral DNA polymerase cofactor pUL44 (19), viral RNA transport factor pUL69 (29), major tegument protein pp65 (51), nuclear egress core protein heterodimer pUL50 -pUL53 (7,27), cellular multiligand binding protein p32/gC1qR (5,19), tumor suppressor protein Rb (9), nuclear lamins A/C (5,28,34,35,38), RNA polymerase II (52), translation factor EF-1␦ (31,32,53), interferon-inducible protein IFI16 (54), and the therapeutically applied nucleoside analog ganciclovir (55,56).…”

Section: Hcmv Protein Kinase Pul97 Interacts With Three Different Typmentioning

confidence: 99%

Cyclins B1, T1, and H differ in their molecular mode of interaction with cytomegalovirus protein kinase pUL97

Steingruber¹,

Keller²,

Socher³

et al. 2019

Journal of Biological Chemistry

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Human cytomegalovirus (HCMV) is a common ␤-herpesvirus causing lifelong latent infections. HCMV replication interferes with cell cycle regulation in host cells because the HCMV-encoded cyclin-dependent kinase (CDK) ortholog pUL97 extensively phosphorylates the checkpoint regulator retinoblastoma protein. pUL97 also interacts with cyclins B1, T1, and H, and recent findings have strongly suggested that these interactions influence pUL97 substrate recognition. Interestingly, here we detected profound mechanistic differences among these pUL97-cyclin interactions. Our study revealed the following. (i) pUL97 interacts with cyclins B1 and H in a manner dependent on pUL97 activity and HCMV-specific cyclin modulation, respectively. (ii) The phosphorylated state of both proteins is an important determinant of the pUL97cyclin B1 interaction. (iii) Activated phospho-Thr-315 cyclin H is up-regulated during HCMV replication. (iv) Thr-315 phosphorylation is independent of intracellular pUL97 or CDK7 activity. (v) pUL97-mediated in vitro phosphorylation is detectable for cyclin B1 but not H. (vi) Mutual transphosphorylation between pUL97 and CDK7 is not detectable, and an MS-based phosphosite analysis indicated that pUL97 might unexpectedly not be phosphorylated in its T-loop. (vii) The binary complexes pUL97-cyclin H and CDK7-cyclin H as well as the ternary complex pUL97-cyclin-H-CDK7 are detectable in an assembly-based CoIP approach. (viii) pUL97 self-interaction can be bridged by the transcriptional cyclins T1 or H but not by the classical cell cycle-regulating B1 cyclin. Combined, our findings unravel a number of cyclin type-specific differences in pUL97 interactions and suggest a multifaceted regulatory impact of cyclins on HCMV replication.

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Rapid detection and quantitation of ganciclovir resistance in cytomegalovirus quasispecies

Cited by 9 publications

References 37 publications

The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting

The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy

Cyclins B1, T1, and H differ in their molecular mode of interaction with cytomegalovirus protein kinase pUL97

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