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KSHV reactivation and novel implications of protein isomerization on lytic switch control.

Guito J, Lukac DM - Viruses (2015)

Bottom Line: In Kaposi's sarcoma-associated herpesvirus (KSHV) oncogenesis, both latency and reactivation are hypothesized to potentiate tumor growth.Dysregulation of Pin1 is implicated in human cancers, and KSHV is the latest virus known to co-opt Pin1 function.Intriguing scenarios for Pin1's underlying activities, and the potential broader significance for isomerization of Rta and reactivation, are highlighted.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School and Graduate School of Biomedical Sciences, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ 07103, USA. ypw1@cdc.gov.

ABSTRACT
In Kaposi's sarcoma-associated herpesvirus (KSHV) oncogenesis, both latency and reactivation are hypothesized to potentiate tumor growth. The KSHV Rta protein is the lytic switch for reactivation. Rta transactivates essential genes via interactions with cofactors such as the cellular RBP-Jk and Oct-1 proteins, and the viral Mta protein. Given that robust viral reactivation would facilitate antiviral responses and culminate in host cell lysis, regulation of Rta's expression and function is a major determinant of the latent-lytic balance and the fate of infected cells. Our lab recently showed that Rta transactivation requires the cellular peptidyl-prolyl cis/trans isomerase Pin1. Our data suggest that proline‑directed phosphorylation regulates Rta by licensing binding to Pin1. Despite Pin1's ability to stimulate Rta transactivation, unchecked Pin1 activity inhibited virus production. Dysregulation of Pin1 is implicated in human cancers, and KSHV is the latest virus known to co-opt Pin1 function. We propose that Pin1 is a molecular timer that can regulate the balance between viral lytic gene expression and host cell lysis. Intriguing scenarios for Pin1's underlying activities, and the potential broader significance for isomerization of Rta and reactivation, are highlighted.

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Rta protein has putative, conserved Pin1 motifs. Blue bars indicate 15 putative S/T-P motifs, sites for Pin1 binding and isomerization, which are characterized by a phosphoserine or phosphothreonine immediately preceding a proline. Letters indicate aa that are parts of possible Pin1 motifs that are phylogenetically conserved between KSHV Rta and at least one additional γ-herpesvirinae family member ORF50 homolog (part of the TAF50 protein superfamily). Motif bars colored in red indicate those that are known to be phosphorylated (T515, S634 and S636; see Figure 4). Putative Pin1 motifs T449, T540, T628, S644 and S650 are also putative phosphorylated residues. Boxes at bottom show alignments of proline-rich and RBP-JK binding regions of Rta. Numbers indicate aa position. +++ = positively-charged aa-rich, LR = leucine heptapeptide repeat domain, S/T = serine/threonine-rich, hyd DE hyd = hydrophobic/charged/hydrophobic aa-rich, NLS = nuclear localization signal sequence, P = conserved proline, S = conserved serine, T = conserved threonine, M = fully conserved S/T-P motif. Figure modified from [166].
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viruses-07-00072-f005: Rta protein has putative, conserved Pin1 motifs. Blue bars indicate 15 putative S/T-P motifs, sites for Pin1 binding and isomerization, which are characterized by a phosphoserine or phosphothreonine immediately preceding a proline. Letters indicate aa that are parts of possible Pin1 motifs that are phylogenetically conserved between KSHV Rta and at least one additional γ-herpesvirinae family member ORF50 homolog (part of the TAF50 protein superfamily). Motif bars colored in red indicate those that are known to be phosphorylated (T515, S634 and S636; see Figure 4). Putative Pin1 motifs T449, T540, T628, S644 and S650 are also putative phosphorylated residues. Boxes at bottom show alignments of proline-rich and RBP-JK binding regions of Rta. Numbers indicate aa position. +++ = positively-charged aa-rich, LR = leucine heptapeptide repeat domain, S/T = serine/threonine-rich, hyd DE hyd = hydrophobic/charged/hydrophobic aa-rich, NLS = nuclear localization signal sequence, P = conserved proline, S = conserved serine, T = conserved threonine, M = fully conserved S/T-P motif. Figure modified from [166].

Mentions: Other possible motifs could also have binding and/or isomerization specificity, including Thr449, Thr515, Ser634 (which is a fully conserved Pin1 motif) and Ser636 (Figure 5) [166]. The latter three, in this case, are interesting putative motifs, as they are the only known potential Pin1 sites to date that has been previously shown to be phosphorylated in in vitro binding assays or in infected cell-based Western blots, by Rta transactivation inhibitor hKFC (for Thr515) and CDK9 (Ser634/636), respectively (and it bears mentioning that the other, putative CDK binding sites, at Thr449, Thr540, Thr628, Ser644 and Ser650, are also all putative Pin1 sites) [42,44,45]. CDK9 kinase activity on Rta is notable because CDK9 is a catalytic subunit of positive transcription elongation factor b (P-TEFb), which associates with the promoter-paused RNAP II’s CTD and activates transcriptional elongation [44]. Rta likely recruits CDK9 to viral promoters, where the kinase licenses transcription as well as positively regulates Rta activity. Pin1, which as previously discussed, binds to the CTD and regulates transcriptional elongation, is known to bind to CDK9-phosphorylated substrate hSpt5, an elongation inhibitor [137,182]. Thus, Pin1 interactions with both CDK9 targets, hSpt5 and Rta, could enhance RNAP II transcriptional elongation at KSHV gene promoters. While it is tempting to suggest that these could represent bona fide pS/T-P motifs, the cytosolic localization of hKFC makes an Rta‑regulating Pin1 modification through hKFC in infected cells suspect, while for CDK9, in light of the lack of Pin1 binding to the Rta aa 525–691 truncation mutant, a true Pin1 motif beyond aa 525 appears less likely [45,166]. Nevertheless, Pin1 binding at Ser634/636 in infected cells, alone or combined, could still be possible, and future functional binding analysis with Pin1 motif mutants of Rta will be required to clarify this interaction. Taking into account the intricate regulation by Pin1 reported throughout this review, multiple binding sites could provide Pin1 with a combinatorial influence on Rta function, much as Pin1 coordinates the CTD code of RNAP II, based directly on the phosphorylation status of certain motifs. This control could be one explanation for the divergent effects of Pin1 during the lytic cycle, which will be further addressed below.


KSHV reactivation and novel implications of protein isomerization on lytic switch control.

Guito J, Lukac DM - Viruses (2015)

Rta protein has putative, conserved Pin1 motifs. Blue bars indicate 15 putative S/T-P motifs, sites for Pin1 binding and isomerization, which are characterized by a phosphoserine or phosphothreonine immediately preceding a proline. Letters indicate aa that are parts of possible Pin1 motifs that are phylogenetically conserved between KSHV Rta and at least one additional γ-herpesvirinae family member ORF50 homolog (part of the TAF50 protein superfamily). Motif bars colored in red indicate those that are known to be phosphorylated (T515, S634 and S636; see Figure 4). Putative Pin1 motifs T449, T540, T628, S644 and S650 are also putative phosphorylated residues. Boxes at bottom show alignments of proline-rich and RBP-JK binding regions of Rta. Numbers indicate aa position. +++ = positively-charged aa-rich, LR = leucine heptapeptide repeat domain, S/T = serine/threonine-rich, hyd DE hyd = hydrophobic/charged/hydrophobic aa-rich, NLS = nuclear localization signal sequence, P = conserved proline, S = conserved serine, T = conserved threonine, M = fully conserved S/T-P motif. Figure modified from [166].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306829&req=5

viruses-07-00072-f005: Rta protein has putative, conserved Pin1 motifs. Blue bars indicate 15 putative S/T-P motifs, sites for Pin1 binding and isomerization, which are characterized by a phosphoserine or phosphothreonine immediately preceding a proline. Letters indicate aa that are parts of possible Pin1 motifs that are phylogenetically conserved between KSHV Rta and at least one additional γ-herpesvirinae family member ORF50 homolog (part of the TAF50 protein superfamily). Motif bars colored in red indicate those that are known to be phosphorylated (T515, S634 and S636; see Figure 4). Putative Pin1 motifs T449, T540, T628, S644 and S650 are also putative phosphorylated residues. Boxes at bottom show alignments of proline-rich and RBP-JK binding regions of Rta. Numbers indicate aa position. +++ = positively-charged aa-rich, LR = leucine heptapeptide repeat domain, S/T = serine/threonine-rich, hyd DE hyd = hydrophobic/charged/hydrophobic aa-rich, NLS = nuclear localization signal sequence, P = conserved proline, S = conserved serine, T = conserved threonine, M = fully conserved S/T-P motif. Figure modified from [166].
Mentions: Other possible motifs could also have binding and/or isomerization specificity, including Thr449, Thr515, Ser634 (which is a fully conserved Pin1 motif) and Ser636 (Figure 5) [166]. The latter three, in this case, are interesting putative motifs, as they are the only known potential Pin1 sites to date that has been previously shown to be phosphorylated in in vitro binding assays or in infected cell-based Western blots, by Rta transactivation inhibitor hKFC (for Thr515) and CDK9 (Ser634/636), respectively (and it bears mentioning that the other, putative CDK binding sites, at Thr449, Thr540, Thr628, Ser644 and Ser650, are also all putative Pin1 sites) [42,44,45]. CDK9 kinase activity on Rta is notable because CDK9 is a catalytic subunit of positive transcription elongation factor b (P-TEFb), which associates with the promoter-paused RNAP II’s CTD and activates transcriptional elongation [44]. Rta likely recruits CDK9 to viral promoters, where the kinase licenses transcription as well as positively regulates Rta activity. Pin1, which as previously discussed, binds to the CTD and regulates transcriptional elongation, is known to bind to CDK9-phosphorylated substrate hSpt5, an elongation inhibitor [137,182]. Thus, Pin1 interactions with both CDK9 targets, hSpt5 and Rta, could enhance RNAP II transcriptional elongation at KSHV gene promoters. While it is tempting to suggest that these could represent bona fide pS/T-P motifs, the cytosolic localization of hKFC makes an Rta‑regulating Pin1 modification through hKFC in infected cells suspect, while for CDK9, in light of the lack of Pin1 binding to the Rta aa 525–691 truncation mutant, a true Pin1 motif beyond aa 525 appears less likely [45,166]. Nevertheless, Pin1 binding at Ser634/636 in infected cells, alone or combined, could still be possible, and future functional binding analysis with Pin1 motif mutants of Rta will be required to clarify this interaction. Taking into account the intricate regulation by Pin1 reported throughout this review, multiple binding sites could provide Pin1 with a combinatorial influence on Rta function, much as Pin1 coordinates the CTD code of RNAP II, based directly on the phosphorylation status of certain motifs. This control could be one explanation for the divergent effects of Pin1 during the lytic cycle, which will be further addressed below.

Bottom Line: In Kaposi's sarcoma-associated herpesvirus (KSHV) oncogenesis, both latency and reactivation are hypothesized to potentiate tumor growth.Dysregulation of Pin1 is implicated in human cancers, and KSHV is the latest virus known to co-opt Pin1 function.Intriguing scenarios for Pin1's underlying activities, and the potential broader significance for isomerization of Rta and reactivation, are highlighted.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School and Graduate School of Biomedical Sciences, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ 07103, USA. ypw1@cdc.gov.

ABSTRACT
In Kaposi's sarcoma-associated herpesvirus (KSHV) oncogenesis, both latency and reactivation are hypothesized to potentiate tumor growth. The KSHV Rta protein is the lytic switch for reactivation. Rta transactivates essential genes via interactions with cofactors such as the cellular RBP-Jk and Oct-1 proteins, and the viral Mta protein. Given that robust viral reactivation would facilitate antiviral responses and culminate in host cell lysis, regulation of Rta's expression and function is a major determinant of the latent-lytic balance and the fate of infected cells. Our lab recently showed that Rta transactivation requires the cellular peptidyl-prolyl cis/trans isomerase Pin1. Our data suggest that proline‑directed phosphorylation regulates Rta by licensing binding to Pin1. Despite Pin1's ability to stimulate Rta transactivation, unchecked Pin1 activity inhibited virus production. Dysregulation of Pin1 is implicated in human cancers, and KSHV is the latest virus known to co-opt Pin1 function. We propose that Pin1 is a molecular timer that can regulate the balance between viral lytic gene expression and host cell lysis. Intriguing scenarios for Pin1's underlying activities, and the potential broader significance for isomerization of Rta and reactivation, are highlighted.

Show MeSH
Related in: MedlinePlus