Atari, N. | bioRxiv Science

bioRxiv2022

P681 mutations within the polybasic motif of spike dictate fusogenicity and syncytia formation of SARS CoV-2 variants

The rapid spread and dominance of the Omicron SARS-CoV-2 over its Delta variant has posed severe global challenges. While extensive research on the role of the Receptor Binding Domain on viral infectivity and vaccine sensitivity has been documented, the role of the spike 681PRRAR/SV687 polybasic motif is less clear. Here we monitored infectivity and vaccine sensitivity of Omicron SARS-CoV-2 pseudovirus against sera samples that were drawn four months post administration of the third dose of BNT162b2 mRNA vaccine. Our findings show that relative to Wuhan-Hu and Delta SARS-CoV-2, Omicron displayed enhanced infectivity and a sharp decline in its sensitivity to vaccine-induced neutralizing antibodies. Furthermore, while the spike proteins form Wuhan-Hu (P681), Omicron (H681) and BA.2 (H681) pseudoviruses modestly promoted cell fusion and syncytia formation, Delta spike (P681R) displayed enhanced fusogenic activity and syncytia formation capability. Live-viruses plaque formation assays confirmed these findings and demonstrated that relatively to the Wuhan-Hu and Omicron SARS-CoV-2, Delta formed more plaques that were smaller in size. Introducing a single P681R point mutation within the Wuhan-Hu spike, or H681R within Omicron spike, restored fusion potential to similar levels observed for Delta spike. Conversely, a R681P point mutation within Delta spike efficiency abolished fusion potential. We conclude that over time, the efficiency of the third dose of the Pfizer vaccine against SARS CoV-2 is waned, and cannot neutralize Omicron. We further verify that the P681 position of the viral spike dictates fusogenicity and syncytia formation.

microbiology↗

bioRxiv2020

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

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 Kaposis 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 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, post-transcriptomic and proteomic landscapes. The ability of KSHV to affect the nucleolus and rRNA modifications constitutes a novel interaction network between viral and cellular components. The study of rRNA modifications is still in its infancy; however, the notion of altering cell fate by regulating rRNA modifications has recently begun to emerge, and its significance in viral infection is intriguing.

microbiology↗