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    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Record number 444709
Title Archaeal virus-host interactions
Author(s) Quax, T.E.F.
Source University. Promotor(en): John van der Oost, co-promotor(en): D. Prangsihvili. - Wageningen : Wageningen UR - ISBN 9789461737830 - 186
Department(s) Microbiological Laboratory
VLAG
Publication type Dissertation, internally prepared
Publication year 2013
Keyword(s) virussen - sulfolobus islandicus rod-shaped virus 2 - archaea - virus-gastheer interacties - infecties - viruses - virus-host interactions - infections
Categories Microbiology (General)
Abstract

The work presented in this thesis provides novel insights in several aspects of the molecular

biology of archaea, bacteria and their viruses.

Three fundamentally different groups of viruses are associated with the three domains of life.

Archaeal viruses are characterized by a particularly high morphological and genetic diversity. Some

archaeal viruses, such as Sulfolobus islandicus rod-shaped virus 2 (SIRV2), have quite remarkable

infection cycles. As described in Chapter 1, infection with SIRV2 results in the formation of

large virus associated pyramids (VAPs) on the host cell surface. The structures open in the final

step of the infection cycle, creating large apertures to release the rod-shaped viruses that have

matured in the cytoplasm. This virus release mechanism is unique and does not resemble egress

mechanisms of bacterial and eukaryotic viruses. Analysis of the protein composition of SIRV2

infected cells, as outlined in Chapter 2, revealed the strong accumulation of the virus encoded

protein PVAP in membranes after infection, suggesting involvement in VAP formation. The

VAPs can be isolated as discrete particles, as demonstrated in Chapter 3. Electron microscopic

survey of these particles showed that they are baseless pyramids with a heptagonal perimeter.

This geometry is exceptional and especially the sevenfold symmetry is very rare in nature

(20S proteasome, myosin). The structures can have various sizes, probably reflecting different

developmental stages. This suggests that they grow by the gradual expansion of the triangular

facets. Analysis of the protein composition of the structures revealed the exclusive presence of

PVAP and anti-bodies raised against this protein labeled specifically the VAPs on thin sections

of infected cells as observed in electron microscopy. PVAP is sufficient for VAP formation, which

was demonstrated by expression of the protein and successful assembly of pyramidal structures,

in the archaeon S. acidocaldarius and the bacteria Escherichia coli. Further analysis of PVAP

truncation mutants as outlined in Chapter 4, showed that besides the 10 C-terminal amino

acids, all domains of the protein are essential for VAP formation. PVAP can form oligomers of

several sizes, including those of a heptamer, which probably act as nucleation points for VAP

formation on the cell membrane. Analysis of the truncation mutants indicated that both the C

and N terminal domain are important for interaction between monomers. Detailed observation

with whole cell cryo-electron tomography of VAPs formed in the natural and heterologous

system, revealed the presence of two layers in the structure. The outer one is continuous with

the cell membrane. The inner layer facing the cytoplasm, presumably represents a protein sheet

formed by tight interactions between the C-terminal domain of PVAP connected with a short

linker region to the membrane. The sheets are slightly bended, giving the complete structure the

appearance of a teepee. At the junction of two triangular sheets, the structure is perforated,

creating predetermined breaking points. Furthermore, in this chapter data is presented which

underlines the unique nature of this protein, since it is able to form VAPs successfully in

archaeal, bacterial and eukaryotic membranes, which all fundamentally differ in protein and

lipid composition. In case of expression in Saccharomyces cerevisiae, VAPs are formed on all

membranes, including those of mitochondria, suggesting that the protein inserts spontaneously

in membranes. Thus, PVAP serves as a universal membrane remodeling system, which might be

exploited for biotechnological purposes, such as the development into a universal system for the

controlled opening of ~100 nm apertures in any lipid bilayer.

Production of VAPs is one of the dramatic consequences that SIRV2 infection has on the

host cell. Whole transcriptome sequencing allowed determination of a global map of virus

and host gene expression during the infection cycle, which is presented in Chapter 5. Directly

after infection, transcription of viral genes starts simultaneously from both genome termini. All

possible protein interactions between all SIRV2 proteins were assayed with yeast two-hybrid

and these results were used to advance current knowledge on SIRV2 genes functions, of which

the majority is still unidentified. The host cells respond to viral infection by adapting expression

of more than 30% of its genes. Genes involved in cell division are down regulated, while those

playing a role in anti-viral defense are activated. Specifically, for the first time massive activation

of toxin anti-toxin and CRISPR-Cas systems is observed in an archaeal system. The different

degree of expression and activation of the various systems highlights the specialized functions

they perform.

The CRISPR-associated multi-subunit ribonucleoprotein complexes that are crucial for the

CRISPR mediated anti-viral defense, generally have an uneven stoichiometry, i.e. the 4-6 different

protein subunits are present in different quantities. Just as most functionally related bacterial and

archaeal genes, the cas genes are clustered in operons, which allow for co-expression (as has

indeed been observed in the transcriptome analysis described in Chapter 5). This is advantageous

when equal amounts of gene products are required, such as is the case for protein complexes

with even stoichiometry. However, a substantial number of important protein complexes

contain uneven stoichiometry. Employing comparative genomics, in Chapter 6, it is shown

that differential translation is a key determinant of modulated expression of genes clustered

in operons and that codon bias generally is the best in silico indicator of unequal protein

production. In addition, analysis of protein production from genes with synonymous mutations

from synthetic operons, provides evidence that initiation of translation can occur at intercistronic

sites. The widespread occurrence of modulation of translation efficiency, suggests that this is

a universal mode of control in bacteria and archaea that allows for differential production of

operon-encoded proteins.

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