Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii.

Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances. Their host, Acanthamoeba castellanii, is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host were lacking. Here, we provide a detailed method for genetic manipulation of A. castellanii and the GVs it plays host to by using CRISPR-Cas9 or homologous recombination. We detail the steps of vector preparation (4 d), transfection of amoeba cells (1 h), infection (1 h), selection (5 d for viruses, 2 weeks for amoebas) and cloning of recombinant viruses (4 d) or amoebas (2 weeks). This procedure takes ~3 weeks or 1 month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which was not possible by using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 h), western blotting (4 h), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 d) and quantification of DNA replication by quantitative PCR (4 h) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen or pathogen-symbiont interactions in A. castellanii, to be studied in vivo.

MMP modulated differentiation of mouse embryonic stem cells on engineered cell derived matrices.

Stem cell differentiation is dictated by the dynamic crosstalk between cells and their underlying extracellular matrix. While the importance of matrix degradation mediated by enzymes such as matrix metalloproteinases (MMPs) in the context of cancer invasion is well established, the role of MMPs in stem cell differentiation remains relatively unexplored. Here we address this question by assaying MMP expression and activity during differentiation of mouse embryonic stem cells (mESCs) on mouse embryonic fibroblast (MEF) derived matrices (MEFDMs) of varying stiffness and composition. We show that mESC differentiation into different germ layers is associated with expression of several MMPs including MMP-11, 2, 17, 25 and 9, with MMP-9 detected in cell secreted media. Different extents of softening of the different MEFDMs led to altered integrin expression, activated distinct mechanotransduction and metabolic pathways, and induced expression of germ layer-specific markers. Inhibition of MMP proteolytic activity by the broad spectrum MMP inhibitor GM6001 led to alterations in germ layer commitment of the differentiating mESCs. Together, our results illustrate the effect of MMPs in regulating mESC differentiation on engineered cell derived matrices and establish MEFDMs as suitable substrates for understanding molecular mechanisms regulating stem cell development and for regenerative medicine applications.

The number of genes encoding repeat domain-containing proteins positively correlates with genome size in amoebal giant viruses.

Curiously, in viruses, the virion volume appears to be predominantly driven by genome length rather than the number of proteins it encodes or geometric constraints. With their large genome and giant particle size, amoebal viruses (AVs) are ideally suited to study the relationship between genome and virion size and explore the role of genome plasticity in their evolutionary success. Different genomic regions of AVs exhibit distinct genealogies. Although the vertically transferred core genes and their functions are universally conserved across the nucleocytoplasmic large DNA virus (NCLDV) families and are essential for their replication, the horizontally acquired genes are variable across families and are lineage-specific. When compared with other giant virus families, we observed a near-linear increase in the number of genes encoding repeat domain-containing proteins (RDCPs) with the increase in the genome size of AVs. From what is known about the functions of RDCPs in bacteria and eukaryotes and their prevalence in the AV genomes, we envisage important roles for RDCPs in the life cycle of AVs, their genome expansion, and plasticity. This observation also supports the evolution of AVs from a smaller viral ancestor by the acquisition of diverse gene families from the environment including RDCPs that might have helped in host adaption.

Genome segregation and packaging machinery in Acanthamoeba polyphaga mimivirus is reminiscent of bacterial apparatus.

UNLABELLED: Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. IMPORTANCE: Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses.