Criss-crossing viruses give rise to peculiar hybrid variants

October 30, 2020

For millions of years, viruses have participated in a far-flung, import-export business, exchanging fragments of themselves with both viral and nonviral agents and acquiring new features. What these tiny entities lack in outward complexity, they make up for with their astonishing abilities to swap out modular genomic components and ceaselessly reinvent themselves.

In new research appearing in the journal mBioArvind Varsani and his colleagues investigate a recently discovered class of viruses that have taken the characteristic versatility of the viral world to new heights. Cruciviruses are a hybrid form containing both RNA and DNA genomic material. Here, a single-stranded DNA virus (yellow) containing a Rep protein sequence, which directs the virus's replication, borrows genetic information from an RNA virus (blue) , specifically, a coding sequence for the RNA virus's capsid protein. The result is a chimerical virus with both DNA and RNA components — a crucivirus (seen in the right panel). Graphic by Shireen Dooling for the Biodesign Institute. Download Full Image

Referred to as cruciviruses, these minute forms reveal a fusion of components from both RNA and DNA viruses, proving that these previously distinct genomic domains can, under proper conditions, intermingle, producing a hybrid or chimeric viral variant.

Varsani, a virologist at the Arizona State Univeristy Biodesign Center for Fundamental and Applied Microbiomics, is deeply intrigued with these new viruses, which are starting to crop up in greater abundance and diversity in a wide range of environments. 

“It is great to see the research groups that first identified cruciviruses around the same time teaming up for the sharing and mining of metagenomic data with an aim to identify a larger diversity of cruciviruses,” said Varsani, an associate professor with the ASU School of Life Sciences.

Arvind Varsani is a virologist with the Biodesign Center for Fundamental and Applied Microbiomics and ASU's School of Life Sciences.

New virus in town

Crucivirus sequences were identified by Varsani’s colleague and co-author Kenneth M. Stedman and his group at Portland State University. The team detected the viruses flourishing in an extreme environment — Boiling Springs Lake (BSL) in Lassen Volcanic National Park,  in northern California. Around the same time, Varsani and Mya Breitbart’s research group identified a crucivirus in a dragonfly sample from Florida.

Since their discovery in 2012, cruciviruses have been found in diverse environments around the world, from lakes in upstate New York and Florida, to the Antarctic and deep-sea sediments. Some 80 distinct cruciviruses had been identified, prior to the current study, which expands the number to 461.

The first cruciviruses were identified using a technique known as viral metagenomics, in which viral genetic material obtained directly from the environment is sequenced rather than being cultivated or cultured from a host species or natural reservoir. 

The results of these early investigations revealed peculiar genetic sequences, radically distinct from anything that had been seen before. These sequences clearly displayed the signature of a DNA virus, yet also contained a gene that appeared to be derived from an RNA virus.

Using a shotgun approach to trawl through a potentially vast sequence space, viral metagenomics enables researchers to identify all of the genomic patterns present in an environmental sample, then separate out distinct viral sequences, like a fisherman retrieving a variety of sea creatures from his net.

The technique has revolutionized the discipline of virology. In addition to identifying a galaxy of previously unknown viruses, metagenomics has offered up exciting clues about genetic diversity and is helping to unlock some of the secrets of viral evolution, all without the need to initially isolate viral species or cultivate viruses in the lab.

Form and function

Cruciviruses belong to a broader class of viruses known as CRESS, (for circular Rep-encoding single-stranded) DNA viruses which have recently been classified into the phylum Cressdnaviricota. The defining characteristic of such viruses is their mode of replication, which relies on a specific component, known as the Rep protein. The Rep protein is important for guiding the replication method of these viruses, known as rolling circle DNA replication. Presence of the Rep protein and rolling circle replication pinpoints a virus as belonging to cressdnaviruses and helps researchers untangle the devilishly complex relationships and lineages found in the viral world.

In addition to the Rep found in cressdnaviruses, cruciviruses contain another centrally important feature — a capsid protein that is similar to that previously found only in RNA viruses. Capsids are vitally important, forming the outer shell or envelope that encloses the virus’s identity — its genetic sequence. The capsid shelters the vital nucleic acids sequestered within from digestion by host cell enzymes, enables virus particles to attach themselves to host cells and allows viruses to evade host cell defenses. Finally, capsids contain specialized features that give the virus its ability to puncture the host cell membrane and inject viral nucleic acid into the cell’s cytoplasm.

Analysis indicates that the capsid protein of cruciviruses is closely related to the capsid protein of another virus from the family Tombusviridae — a single-stranded RNA virus known to infect plants. This hybrid viral character, containing both DNA- and RNA-virus-derived coding components, is what makes cruciviruses so unique.

Uncertain origins

But how did a run-of-the-mill cressdnavirus come to acquire its RNA virus capsid protein coding sequence? This remains an issue of considerable debate, though presumably some form of lateral gene transfer occurred.

Viruses can acquire genes from their immediate progenitors, the way genetic traits are passed from human parents to their offspring. Viruses, however, are far more genetically promiscuous, collecting new genes from the cells they infect, from other unrelated viruses and even from bacterial symbionts. (The phenomenon is also common among bacteria, which can use horizontal gene transfer to acquire antibiotic resistance.) 

Through some such mechanism, a cressdnavirus acquired an RNA virus capsid-like gene, creating the first crucivirus. It also appears that various cruciviruses have actively exchanged functional elements among themselves, further scrambling their evolutionary history.

While the HOW of crucivirus DNA-RNA recombination remains mysterious, the WHY may be more straightforward. Clearly, the ability to borrow genetic traits from such distantly related viral sources could provide single-stranded DNA viruses with a considerable adaptive edge.

Collector’s edition

In the current study, researchers explored a vast dataset including 461 cruciviruses and 10 capsid-encoding circular genetic elements identified from varied environments and organisms, making this the most expansive investigation of crucivirus sequences yet undertaken.

The samples were found in environments ranging from temperate lakes to permafrost and lurking within organisms including red algae and invertebrates. The study points to the stramenopiles/alveolates/Rhizaria or SAR supergroup, (a diverse assemblage of eukaryotes, including many photosynthetic organisms) as the plausible candidate hosts for these unusual viruses, though this has yet to be verified. 

After examining the windfall of sequences, the researchers assembled similarity networks of cruciviral proteins with related viruses to try to better understand the twisting evolutionary paths that may have given rise to them, finding a rich cross-pollination of viral traits between many large families of viruses including Geminiviridae, Circoviridae, Nanoviridae, Alphasatellitidae, Genomoviridae, Bacilladnaviridae, Smacoviridae and Redondoviridae.

Intriguing questions remain

The findings may provide new insights into the early transition from RNA as the primary hereditary molecule of life to the adoption of more complex DNA genomes that has come to dominate life in the cellular world. The existence and behavior of cruciviruses suggest that viruses may have played a crucial role in this all-important transition, acting as a kind of genomic bridge between the RNA and DNA worlds, during the earliest emergence of life, though much more work is needed to explore these possibilities.

Recombining in endless forms, viruses have become the planet’s most ubiquitous biological entities, affecting every living organism and occupying every ecological niche. Increasingly, viruses are revealing themselves not only as agents of disease but as drivers of species evolution and vital actors in the molding of ecosystems.

The expanded abilities of cruciviruses to borrow genomic elements from the most far-flung regions of viral sequence space suggest that entirely new virus groups may arise though prolific recombination events between distantly related forms.

Richard Harth

Science writer, Biodesign Institute at ASU


Digging into claws: ASU student helps discover the fossil of a new dinosaur-like species

October 30, 2020

Xavier Jenkins’ alarm goes off at 5:30 a.m. on a Saturday morning. The Arizona State University student and his roommate Ben Kligman quickly get ready and head to the dining hall to stock up on essential supplies for the day — granola bars, lunch meat, four liters of water and most importantly, black coffee. Along with other interns, they stop by the paleontology lab to pack their field gear — hand tools, plaster and other excavation equipment. 

There are seven people headed to the dig site. It’s almost an hourlong car ride along a dirt road to their destination: Thunderstorm Ridge, located in Petrified National Forest. This fossil excavation site was discovered a few years ago. Made up of soft gray dirt and rocks, it’s rich in tiny micro invertebrate fossils that lived in a tropical or semitropical climate approximately 220 million years ago. Xavier Jenkins with Coprolite Xavier Jenkins holding coprolite (fossilized dung) at Thunderstorm Ridge, Petrified Forest National Park in 2019. Photograph courtesy of Xavier Jenkins. Download Full Image

The interns, researchers and park staff spend the whole day out in the field, gathering material to be analyzed the same day at the on-site lab. Jenkins appreciates the rarity of this luxury, noting how paleontologists often wait weeks or even months to properly process their field material.

Back at the lab, Kligman and Jenkins work together analyzing what was brought in from the field. At Thunderstorm Ridge, they collect sections of rock and sediment, because many fossils are too small to see with the naked eye. Jenkins analyzes the material under a microscope. 

Kligman excitedly rushes over to show Jenkins a “big” claw mixed in with the material brought in from the field. The 2-centimeter claw is larger than most fossils found at this site — even bigger than jawbones of some of the other species uncovered at Thunderstorm Ridge.

Equipped with a solid understanding of dinosaur bones, both Jenkins and Kligman knew instantly that this claw belonged to a member of the drepanosaurid family, but they didn’t know it belonged to a previously undiscovered species.

Jenkins had helped discover this new species of dinosaur-like creature. The species is now called Skybalonyx skapter and was small, about a foot long, with a special claw that distinguishes it from other species in the drepanosaurid family. The discovery and analysis was published in October in the Journal of Vertebrate Paleontology.

Jenkins graduated from ASU in May 2020, with Bachelor of Science degrees in anthropology from the School of Human Evolution and Social Change and biological sciences from the School of Life Sciences. The summer after his junior year, Jenkins completed a 12-week paleontology internship at Petrified Forest National Park.

“Even though living on-site was pretty isolating, almost every week a new university came in to conduct research,” Jenkins said. “Big-name paleontology professors visited to work on their own research, and I could help them and network the entire time.” 

Petrified Forest National Park 2019

Field crew excavating at Thunderstorm Ridge, Petrified Forest National Park in 2019. Photograph courtesy of Xavier Jenkins.

The research continued

Petrified Forest paleontologists told Jenkins the newly discovered creature was obviously burrowing, because of the shape of the claw and other fossils identified at the site. But Jenkins wanted to better understand how and why the Skybalonyx skapter would have been a burrowing species, because other members of the drepanosaurid family have been classified as tree-climbing.

The Skybalonyx skapter had a special claw. In scientific terms, it’s called a disarticulated manual ungual. Jenkins wanted to compare the Skybalonyx skapter claw to the claws of different animals alive today.

Having worked previously in the lab with School of Human Evolution and Social Change President’s Professor Kaye Reed, Jenkins approached her with an idea to gather comparative data. Reed helped secure undergraduate research funding to send Jenkins to Washington, D.C., to conduct the research. 

Jenkins visited bone collections at the Museum of Comparative Zoology at Harvard University and the Smithsonian to measure the claws of almost 200 species, including squirrels, chameleons and sloths. He measured the width, length and height of claws from reptiles and mammals, and creatures that climbed trees, dug burrows and lived underground. 

The findings indicated that Skybalonyx skapter’s special claw most closely resembled the claws of burrowing animals like echidnas, moles and tortoises.

“Xavier developed this research on his own — he outlined the project and we discussed methodologies,” Reed said. “It was so exciting when he ran the multivariate analysis and categorized groups of animals based on how they used their manual digits. Xavier was tenacious throughout the process, and an exemplary undergraduate student. He’s going to be an excellent paleontologist.”

Skybalonyx Skapter artwork by Midiaou Diallo

Artist Midiaou Diallo’s depiction of Skybalonyx skapter emerging from a burrow.

A young start in paleontology

“I was raised on dinosaur books,” Jenkins said. “My parents fed me Richard Dawkins' biology books since I was a kid, because that's what I expressed interest in, and I've been following that path ever since then.” 

Through high school and undergraduate studies at ASU, Jenkins was ambitious, seeking out opportunities to learn more about studying dinosaurs as a career. He participated in fieldwork during high school, attended a vertebrate paleontology conference, helped faculty with lab research during college and was a member of ASU’s Undergraduate Anthropology Association.

“I didn't know what I wanted to do in paleontology, but I knew I wanted to be out there, digging,” Jenkins said.

Advice to current students

Jenkins took advantage of unique opportunities available at ASU. “I'm a strong proponent of undergraduate research,” Jenkins said. “This research wouldn’t have been possible if not for working in Kaye Reed’s lab. I also worked in Curtis Marean's lab for a year and that was great.”

Working in these labs was not a part of a posted internship. Instead, Jenkins simply asked if they needed help. He had already secured their permission to take graduate-level courses as an undergraduate student.

To students worried about reaching out to a professor, Jenkins recommends just asking. “Professors are people too,” he said. “And they're often doing such prolific work — especially at (the School of Human Evolution and Social Change). They can be on dozens of research projects and could use your help for something.”

What’s next

Jenkins is now working toward a PhD in biological sciences at another prestigious university. After earning a doctorate, he would like to go straight into a postdoc program, and someday have his own research lab as a tenured professor.

He wants to continue to examine the role of specific dinosaurs as individuals within an ecosystem. This study can be described as paleoecology. “I think it's great to find new species and describe them,” Jenkins said. "But if you're not understanding what they were actually doing, because they were living and breathing animals, what's the point?”

Taylor Woods

Communications program coordinator, School of Human Evolution and Social Change