Dual-RNAseq Analysis Unravels Virus-Host Interactions of MetSV and Methanosarcina mazei

Abstract

Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (TEM). The transcriptome analysis strongly indicates a combined role of virus and host genes in replication, virus assembly, and lysis. Thereby, 285 host and virus genes were significantly regulated. Within these 285 regulated genes, a network of the viral polymerase, MetSVORF6, MetSVORF5, MetSVORF2, and the host genes encoding NrdD, NrdG, a CDC48 family protein, and a SSB protein with a role in viral replication was postulated. Ultrastructural analysis at 180 min p.i. revealed many infected cells with virus particles randomly scattered throughout the cytoplasm or attached at the cell surface, and membrane fragments indicating cell lysis. Dual-RNAseq and qRT-PCR analyses suggested a multifactorial lysis reaction in potential connection to the regulation of a cysteine proteinase, a pirin-like protein and a HicB-solo protein. Our study’s results led to the first preliminary infection model of MetSV infecting M. mazei, summarizing the key infection steps as follows: replication, assembly, and host cell lysis.

Archaeal virus entry and egress

“…Most identified viral receptors stem from Sulfolobales and haloarchaea. One exception is the methanogenic archaeal virus Methanosarcina spherical virus (MetSV), which attaches to its host Methanosarcina mazei via the S-layer or S-layer-associated proteins (Weidenbach et al 2017 , Gehlert et al 2022 ).…”

“…The tailless Enterobacteria phage PRD1 (PRD1) forms a membrane tube from its internal membrane vesicle, along which a linear dsDNA genome can be injected into the host cytoplasm (Peralta et al 2013 ). Since the archaeal viruses SH1, HHIV-2, HCIV-1, and MetSV share a similar virion architecture, with an internal lipid membrane and a linear dsDNA genome, they conceivably could follow the same genome delivery strategy as PRD1 for genome delivery (Bamford et al 2005 , Gehlert et al 2022 ). For STIV, the domain 2 of the turret protein C381 shows highest sequence similarity to the knob domain of the podophage HS1 needle tip, which is likely involved in DNA ejection.…”

In vivostructure probing of RNA inArchaea: Novel insights into the ribosome structure ofMethanosarcina acetivorans

“…These investigations have revealed the nature of RNA structure under diverse growth conditions including unstressed (Ding et al 2014), amino acid-stressed (Ritchey et al 2020), heat-shocked (Su et al 2018) heat-varied (Jolley et al), and salt-stressed growth conditions (Tack et al 2020). In addition to the extensive research on eukaryotic and bacterial systems, the archaea domain of life has been explored with respect to RNA half-life, abundance, and in vivo interactions (Peterson et al 2016;Knüppel et al 2020;Gehlert et al 2022). Nonetheless, methods for probing RNA structure have not yet been widely adapted or applied to archaea.…”

Newly Established Genetic System for Functional Analysis of MetSV

“…MetSV, belonging to the Tectiviridae, infects M. mazei strains with a narrow host range [35,37]. When MetSV was first described in 2017, its linear genome of roughly 10 kb was predicted to encode 22 ORFs, all on one strand with the same direction of transcription and with a mostly unknown function [35,36]. In addition to the polymerase of type B, a potential ATPase and its capsid proteins, no functions could be assigned to the predicted proteins [35].…”

“…Consequently, nine viruses or virus-like particles have been described to infect methanoarchaea to date [ 1 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. Within this small group of viruses, only two are infecting Methanosarcina species, Methanosarcina virus (MetMV) [ 29 ] and Methanosarcina spherical virus (MetSV) [ 35 , 36 ]. While MetMV was detected based on metagenomic approaches, MetSV was isolated from a waste-water treatment plant and can be propagated in the laboratory.…”