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Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38527209
202402541
10.1073/pnas.2402541121
commCommentarymicrobioMicrobiology423
437
Commentary
Biological Sciences
Microbiology
March of the proviruses
Barth Zachary K. a
Aylward Frank O. faylward@vt.edu
a b 1 https://orcid.org/0000-0002-1279-4050

aDepartment of Biological Sciences, Virginia Tech, Blacksburg, VA 24061
bCenter for Emerging, Zoonotic, and Arthropod-Borne Pathogens, Virginia Tech, Blacksburg, VA 24061
1To whom correspondence may be addressed. Email: faylward@vt.edu.
25 3 2024
2 4 2024
25 9 2024
121 14 e2402541121Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
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pmcProviruses, viruses integrated within cellular genomes, are known to be major components of eukaryotic genomes. Within the human genome alone, an estimated 8% of sequence belongs to retroviruses (1). While the presence of these retroelements has long been appreciated, a growing body of literature has recently shown that DNA viruses large and small are also common constituents of eukaryotic genomes. A diverse repertoire of DNA viruses can be found integrated within eukaryotic germlines, including herpesviruses in some humans (2) and fish (3), giant viruses in protists (4), and smaller (~10 to 30 kb) dsDNA viruses in protists and animals (5, 6).

While recent genomic studies have shown that endogenous DNA viruses are abundant in eukaryotic genomes, relatively few studies have demonstrated their reactivation, leaving open the question of whether many of these proviruses are functional or merely genomic relics. In this issue, Koslová et al. (7) demonstrate the reactivation, mobilization, and integration of several endogenous mavirus-like elements (EMALEs) found within the marine unicellular flagellate Cafeteria burkhardae. Notably, active EMALEs were detected in the Atlantic Ocean, Pacific Ocean, and Baltic Sea, suggesting that these integrative viruses have a global reach.

Perhaps the most interesting aspect of EMALE biology is their choice of prey. While the EMALEs integrate within cellular genomes, they are actually virophages, parasites of giant viruses that infect and kill the virophages’ host cells (8). Koslová et al. show that these new EMALEs, like their original mavirus namesake (9), parasitize the giant virus CroV. This parasitism acts as a kind of antiviral immunity and is actually protective toward Cafeteria populations. The reproduction and spread of CroV are limited enough by EMALE propagation to greatly reduce mortality of experimental Cafeteria populations. This protective phenotype is well described in virophages (9, 10), and the integration and reactivation of Mavirus has previously been shown (9); however, Koslová et al.’s work is the first to show that naturally occurring endogenous virophages are protective toward their host organisms. This lends considerable weight to the ‘virophage defense’ hypothesis that posits that virophage proviruses are maintained within host cells as a defense against predatory viruses.

Among eukaryotes, virophage defense against predatory viruses has only been observed in single-celled protists; however, intriguing parallels have been seen in the bacterial world. Recent work has uncovered a staggering number of bacteriophage satellites (11, 12), mobile elements that rely on bacteriophages for their mobilization. Most of these elements are technically not viruses because they redirect the capsid proteins encoded by their host viruses to their own genome rather than encoding capsid proteins of their own. These elements do possess dsDNA genomes of a similar size to virophages, and their parasitism often impairs a reproductive burden on their host virus. In multiple cases, this burden on viral reproduction has a protective effect for the bacterial population (13). This protective phenotype is especially heightened and well explored in the Phage inducible chromosomal island-like elements (PLEs) found in the bacterium Vibrio cholerae (14). Different V. cholerae strains can encode PLE variants that provide protection against the ICP1 bacteriophage. Certain PLEs are able to completely restrict reproduction of certain ICP1 isolates, and ICP1 has in turn evolved a number of specific anti-PLE genes and defense systems (15, 16) that are subject to selective sweeps along with the PLE genes they target (17). A similar arms race is likely occurring between giant viruses and virophages. Koslova et al. show that most EMALEs are not induced in response to CroV, and the ones that are induced exhibit wide variation in their reproductive efficiency and ability to restrict CroV infections.

An important future direction of this work will involve probing the molecular underpinnings of these distinct EMALE phenotypes, and Koslova’s establishment of isogenic EMALE variant strains is a critical first step in understanding the giant virus-virophage arms race. The diversity of endogenous virophages in a protist genome can be quite high, and use of these isogenic strains will enable future work that disentangles the phenotypic consequences of EMALE diversity. Interestingly, most endogenous virophages in C. burkhardae are not induced upon CroV infection, suggesting that they may offer protection against other giant viruses. This is certainly plausible given the enormous diversity of giant viruses in the environment (4), and it further suggests that endogenous virophages offer a kind of genomic palimpsest of past and present inter-viral conflicts.

It is striking that viral defense provided by the parasitism of endogenous viruses or virus-like elements has evolved several times independently in different domains of life. This development parallels a series of recent discoveries that many intracellular anti-viral defense systems originally found in animals belong to highly diverse families that are well represented bacteria (18, 19). Thus, the methods of anti-viral defense appear to be more universal than has historically been assumed. It Is tempting to speculate that endogenous viruses might even play a protective role for animals. Small dsDNA proviruses are abundant in certain animal lineages, such as bony fish (20), and recent work in insects has implicated similar dsDNA viruses in the parasitism of baculoviruses (21) and poxviruses (22). The study of eukaryotic dsDNA proviruses is a quickly developing field and one that has strong implications for our understanding of viral immunity and eukaryotic genome evolution.

Kosolová et al.’s work is the first to show that naturally occurring endogenous virophages are protective towards their host organisms.

Author contributions

Z.K.B. and F.O.A. wrote the paper.

Competing interests

The authors declare no competing interest.

See companion article, “Endogenous virophages are active and mitigate giant virus infection in the marine protist Cafeteria burkhardae,” 10.1073/pnas.2314606121.
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