Lytic Reactivation of the Kaposi’s sarcoma-associated herpesvirus (KSHV) is Accompanied by Major Nucleolar Alterations

Abstract

The nucleolus is a sub-nuclear compartment whose primary function is the biogenesis of ribosomal subunits. Certain viral infections affect the morphology and composition of the nucleolar compartment and influence ribosomal RNA (rRNA) transcription and maturation. However, no description of nucleolar morphology and function during infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is available to date. Using immunofluorescence microscopy, we documented extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. This was manifested by redistribution of key nucleolar proteins, including the rRNA transcription factor, UBF, the essential pre-rRNA processing factor Fibrillarin, and the nucleolar multifunctional phosphoproteins Nucleophosmin (NPM1) and Nucleolin. Distinct delocalization patterns were evident; certain nucleolar proteins remained together whereas others dissociated, implying that nucleolar proteins undergo nonrandom programmed dispersion. Of note, neither Fibrillarin nor UBF colocalized with promyelocytic leukemia (PML) nuclear bodies or with the viral protein LANA-1, and their redistribution was not dependent on viral DNA replication or late viral gene expression. No significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found by RT-qPCR and Northern blot analysis, respectively. Furthermore, fluorescent in situ hybridization (FISH), combined with immunofluorescence, revealed an overlap between Fibrillarin and internal transcribed spacer 1 (ITS1), which represents the primary product of the pre-rRNA, suggesting that the processing of rRNA proceeds during lytic reactivation. Finally, small changes in the levels of pseudouridylation were documented across the rRNA. Taken together, our results suggest that rRNA transcription and processing persist during lytic reactivation of KSHV, yet they may become uncoupled. Whether the observed nucleolar alterations favor productive infection or signify cellular anti-viral responses remains to be determined. Author SummaryWe describe the extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV. Distinct delocalization patterns are illustrated: certain nucleolar proteins remained associated with each other whereas others dissociated, implying that nucleolar proteins undergo nonrandom , 3 programmed dispersion. Of note, no significant changes in pre-rRNA levels and no accumulation of pre-rRNA intermediates were found, suggesting that pre-RNA transcription and processing continue and could be uncoupled during lytic reactivation. Small changes in the levels of pseudouridylation were documented across the rRNA. Previous studies showed that the different forms of KSHV infection are controlled through cellular and viral functions, which reprogram host epigenetic, transcriptomic, posttranscriptomic and proteomic landscapes. The ability of KSHV to affect the nucleolus and rRNA modifications constitutes a novel interaction network between viral and cellula...

The KSHV ORF20 Protein Interacts with the Viral Processivity Factor ORF59 and Promotes Viral Reactivation

“…In addition, nucleolin (NCL) was identified to be in close proximity to ORF20, which is intriguing, since ORF20 ortholog UL24 in HSV-1 was shown to be directly involved in the dispersal of NCL during infection ( 29 ). This suggests that this dispersal, which is also observed during KSHV infection ( 36 , 37 ), might be a conserved function of ORF20.…”

A Non-Coding RNA Network Involved in KSHV Tumorigenesis

“…In the latter Proteasome subunit protein PSMD1 , nucleoporins NUP50 , NUP153 , nucleolar protein NPM1, or exportin 1 XPO1 have been shown to modulate HIV infection or other viral cycles ( 55 – 58 ). In addition, in a preprint article it was postulated an extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV, with redistribution or degradation of proteins such as NPM1 ( 59 ). More interestingly NPM1 (aka NPM) is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.…”

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“…In the latter Proteasome subunit protein PSMD1 , nucleoporins NUP50, NUP153 , nucleolar protein NPM1 or exportin 1 XPO1 have been shown to modulate HIV infection or other viral cycles [Gadad et al, 2011; Behrens et al, 2017; Kane et al, 2018; Rathore et al, 2020]. In addition, in a preprint article it was postulated an extensive destruction of the nuclear and nucleolar architecture during lytic reactivation of KSHV, with redistribution or degradation of proteins such as NPM1 [Atari et al, 2020]. More interestingly NPM1 (aka NPM) is a critical regulator of KSHV latency via functional interactions with v-cyclin and LANA.…”