Giant Soil Viruses

by Abigail Curtis
 

My first niche microbiological interest came fresh­man year of high school, when I dis­cov­ered that viruses don't solely infect ani­mals. Indeed, viruses are per­vasive in every eco­system, inc­luding the small­est ones in the ground. The aspect of viruses in microb­ial eco­sys­tems that I found most fas­cin­ating was the con­cept of giant viruses, and the enig­matic char­acter of their genome and impor­tance. Ever since, I've tried my best to keep up to date on the sub­ject. I find the recent turn to sequen­cing tech­nol­ogies as an ap­proach to the dis­cov­ery of the world of viruses below our feet incred­ibly excit­ing.
 

Figure 1. The giant virus, Pithovirus sibericum ex­t­ract­ed from per­ma­frost in 2014. Source. Fron­tispiece: soda lake Tupanvirus. Source

The genus Mimiviridae con­tains some of the lar­gest viruses known to human­kind. In fact, a 2010 STC post by Merry Youle high­lighted the dis­cov­ery of Acanth­amoeba polyphaga mimi­virus (APMV), shatter­ing prev­ious records with a gen­omic size of ~1.2 Mbp and capsid size of ~500 nm. However, since the time that post – "A Giant Among Giants" – ap­peared, there have been inc­reased efforts and im­proved tech­nologies in the mission to dis­cover and map the viral com­posi­tion of ter­rest­rial en­viron­ments.
 

No longer are scien­tists req­uired to cul­tivate viruses in hosts. The combin­ation of next gen­era­tion seq­uen­cing tech­nol­ogies, open access seq­uence data­bases, and pro­grams that speci­fically mine data­bases to iden­tify viral gen­omes, such as VirSorter and VirFinder, now permit the quick identi­fication of viral gen­omes. Of note is a new ap­proach ref­erred to as "mini-meta­gen­omics." Rather than isolate genetic mat­erial from all of the microbes within a sample, a mini-meta­gen­omic ap­proach first sorts microbes into smaller sub samples through parallel­iza­tion, a pro­cess carried out using micro­fluidic dev­ices. Samples that contain in the order of 100 microbes serve to generate the "mini-meta­gen­omic" seq­uences. This ap­proach is best for ana­lyz­ing small samples with high micro­bial div­ersity, as it prov­ides meta­genome-assembled gen­omes (MAGs) with a greater degree of accur­acy. With the dev­elop­ment of both bulk meta­gen­omics (which yield "bulk MAGs") and mini-meta­gen­omics (which yield "sorted-MAGs"), inves­tigators can now dis­cover more viruses with­out hav­ing to know their host.
 

Using both ap­proaches, a group study­ing soil sam­ples from Harvard Forest dis­cov­ered six­teen new soil giant viruses. Mini-meta­gen­omics yielded 15 gen­omes, while bulk meta­gen­omics yielded only one. In addi­tion, the higher qual­ity seq­uences ob­tained from mini-meta­gen­omics allowed for more accur­ate virus classi­fica­tion. The pres­ence of hall­mark genes encod­ing the major cap­sid pro­tein (MCP), para­log­ous genes, and pack­aging ATPases al­lowed them to unambig­uously clas­sify the new viruses as nucleo­cyto­plasmic large DNA viruses (NCLDVs). The authors were able to place 10 of their 16 viral gen­omes within the genus Mimiviridae. Three gen­omes seemed to form a new group, while the rem­ain­ing gen­omes were not com­plete enough to allow classi­fica­tion.
 

Figure 2. Comparison of mini and bulk meta­gen­om­ics. Source.

New seq­uen­cing tech­nol­ogies have not only en­hanced abil­ities to dis­cover new viruses. They also pro­vide new knowl­edge reg­arding the role and impor­tance of giant viruses in their environ­ments, along with con­firm­ing the rad­ical nat­ure of their gen­omes. In a study analyz­ing 501 NCLDV MAGs, viruses within Mimiviridae and Phycodnaviridaefamilies contain genes en­coding enzymes essen­tial for glycol­ysis and the citric acid cycle. Most prev­alently among them, the glyco­lytic glycer­aldehyde-3-phos­phate dehydro­genase (G3P), phospho­glycer­ate mutase (PGM), phospho­glycer­ate kinase (PGK), and the tri­carbox­ylic aconit­ase and suc­cinate dehydro­genase (SDH). One MAG had 7 out of 10 genes neces­sary for glycol­ysis. These find­ings indicate giant viruses' extra­ordin­ary gen­omic indep­endence from their hosts (termed “quasi-auton­omous” viruses) and high­lights a poten­tial for patho­gens to recon­struct metab­olism in viro­cells.
 

Another unique giant virus is the tupan­virus, found in oceanic and lake sedi­ments, which con­tains the most compre­hensive set of genes involved in trans­lation, including genes for 20 amino­acyl tRNA synthe­tases, 11 trans­crip­tion fac­tors, and up to 70 tRNAs in one gen­ome. The 'soda lake' strain of Tupan­virus is also unique amongst giant vir­uses in its abil­ity to infect sev­­eral protist hosts, such as sev­eral Acanthameoba species, and num­erous phyto­plankton strains.
 

Giant viruses continue to excite and intrigue, as mem­bers contra­dict another uni­versal charac­teristic of viruses: capsid pro­teins. While the known mem­bers of the Mimiviridae family have capsid shells in nearly a perfect icosa­hedral shape, there are not­able giant viruses that do not. Pandora­viruses, Cedratviruses, and Orpheoviruses lack a capsid gene; in its place a tegument-adjacent en­velope. The Pithoviruses stand out because, al­though they con­tain genes coding for a capsid pro­tein, do not present one pheno­typ­ically. In contrast, fausto­viruses have two protein shells, the outer­most layer con­sisting of a capsid pro­tein found in clas­sical viruses, while the inner casing is hexa­meric.
 

Figure 3. Faustovirus cross sections high­light­ing (C) in­ner and out­er pro­tein shells, (D) icosa­hed­ral shape sha­ded by rad­ius. Scale bars: 50µm. Source.

All the viruses afore­men­tioned were discov­ered in the past 15 years, sugges­ting that scien­tists have barely begun to under­stand Earth's viral diver­sity. Since viral bio­diver­sity is incred­ibly vast, and the over­lap of genes between cur­rently known species is slim, cons­truct­ing a compre­hensive phylo­genetic tree remains dif­ficult. Yet, theories abound regard­ing their evolu­tion­ary origins. One theory is that giant viruses formed before the ribo­some for­mation, but after the evol­ution of amino­acyl tRNA synthe­tases, and they have since ac­quired meta­bolic genes from hosts via lat­eral gene trans­fer.
 

Megavirales'  challenge against pre-exist­ing classi­fica­tions of microbes has led scien­tists to think out­side of the box, result­ing in the term “Things Resist­ing Uncom­pleted Classi­fica­tions”(TRUC). Form­ing a fourth branch of the microb­ial world, TRUC consist­s of microbes defy­ing tradi­tional classi­fica­tion. Within this cate­gory, Megavirales, Pandoraviruses, and other giant viruses form a "super­group" due to their unusual mix­ture of gen­omic and pheno­typic features.
 

Figure 4. Predicted system of virus-host int­er­ac­tions in the rhizo­sphere, in­di­ca­ting areas of fut­ure in­ter­ests in study­ing viral func­tion in soil micro­bi­omes. Source.

But why should we care about viruses from soil? We don’t live in the soil, and soil viruses are not likely to cause infec­tion in humans. Still, these crea­tures play impor­tant roles in soil ecology, exemplified in carbon cyc­lings. In the perma­frost thaw grad­ient of north­ern Sweden, Trouble et al. (2018), util­ized viral opera­tional taxon­omic units (vOTUs), find­ing link­ages to lin­eages of Acidobacteria, Deltaproteobacteria, and Verrucomicrobia that resulted in the asso­ciation of 30 auxil­iary meta­bolic genes (AMGs) pres­umed to play a role in carbon metab­olism. More rec­ently, data from soil samples from Santa Fe and Pajarito (both in New Mexico), suggests that inc­reased bac­terial abun­dance can, via alter­ing dyn­amics between fellow microbes, change both carbon and nut­rient cyc­ling. Although terres­trial micro­biomes do not share "the viral shunt" of their aquatic counter­parts, viral infec­tion (caused by RNA viruses) trig­gering apop­tosis is hypoth­esized to mobil­ize cell carbon that plays a vital role in carbon cyc­ling. Without soil viruses, the deli­cate eco­system upon which much of our food supply rests would det­erior­ate. With tech­nol­ogies like mini-meta­gen­omics, the contin­ued identi­fic­ation of new viruses and inc­reased under­stand­ing of viral func­tion and phylo­geny, advances in the field of soil micro­biology can not only be hoped for, but expec­ted.
 

 
 

Abby is an incoming first-year student at Harvard College with an intended joint concentration in molecular and cellular biology and physics; post-grad, she hopes to become a microbiologist. She is currently a summer researcher in the Isberg lab at Tufts Medical School, working with Legionella pneumophila.