On the experimental generation of endogenous (non-retroviral) RNA viruses

A retrovirus. http://www.itqb.unl.pt/

The sheer amount of genomic data now available from a wide range of species has allowed the increased scrutiny over what genes and DNA sequences are present in their chromosomes. What we have begun to notice is that many of these sequences have a viral origin. 

And, in the recent half-decade, the numbers of these endogenous viruses discovered have rapidly increased, but how did they get there? What are they doing? And, are they bad for us? Only a true experimental model system can answer these question but this is something which is lacking.

Lets talk about ERVs

Now, viruses have left their mark on our genomes in more ways than one; infection and associated disease/mortality has heavily influenced the genetic structure of populations via natural selection and genetic drift for millions of years. Yet, another important mechanism is that employed by the endogenous retroviruses (ERVs) that have inserted a DNA copy of themselves into our chromosomes - the norm for retroviruses - and have forever become part of us.

Over the course of evolution, these once infectious viruses have become redundant, building up a collection of genetic mutations resulting in loss of replicative ability. Although many still play a role in the cellular biology of the host and have been a great source of genetic novelty over the billions of years of evolution.


For some excellent info on these viruses, see ERVs archive of ERV-related material.


What about non-ERVs?

However, what we have noticed is that viruses other than retroviral species have inserted themselves into genomes of humans, other animals and even plants and fungi. Many of these viruses have a DNA phase in their replication cycle, which is put into the genome of their host to aid their survival and so it may not be all that surprising that they have stayed with us through evolution (these viruses include many single-stranded DNA viruses and again).

One intriguing observation is that many of these non-retroviral endogenous viruses are in fact - or were - RNA viruses with no known DNA phase during replication. They are therefore called Non-retroviral RNA virus sequences (NRVSs). See plant NRVSs and mammalian NRVS (ebola virus-like , borna virus-like  and many more - (lots, they're everywhere). There is strong evidence that these integrations occurred thousands, if not millions of years ago and could have played a role in the evolution of many species.


How can we study these viruses?

But just exactly how do these viruses do it? After-all, they are RNA viruses without a reverse-transcriptase enzyme and hence no natural ability to produce a DNA genome that can be inserted into our chromosomes. And, can we follow this endogenisation experimentally? One mechanism is thought to occur when an endogenous retrovirus-like element joins itself to a non-endogenous RNA virus and then this chimera is put into our genome. But this is really only half the story - can we ever study the entire process, from initial infection to endogenisation?

 For an RNA virus to become fully integrated into our germline it has to first infect our germ-line cells (sperm/oocytes); its RNA genome must be copied into DNA and this DNA molecule must be inserted into the chromosome. It also must allow for the development of healthy and reproductively active offspring and can then let evolution take its course. An experimental model system of this process would allow for a better understanding of this process in molecular detail and how this relates to the evolutionary process as a whole.

Here's how you would do it:

The animal model

Bank vole - a good model for endogenous viruses?

A small-animal model that could be infected by a  type of virus that had been shown to integrate into the genome (borna disease virus, for example) would make this easier to study. Plus, many rodents have been shown to harbour many NRVSs already.

The virus infection

You would infect the animals with the virus in as natural conditions as possible and look to see whether the virus entered and replicated in the cells of the germ-line.A GFP-expressing virus would work best for this.

Detection of RNA - DNA

What you would have to do is be able to track the process of turning the RNA genome into DNA. A PCR-based screening would work well for this and could be applied to a range of tissues in the host, including occytes/spermatozoa.

Integration

To prove that the DNA copy was inserted into the host chromosome you would need to sequence the sites where the DNA had integrated in and determine where in the genome it lay.

Stability

This experimentally infected rodents could be bred continously and the presence of endogenised virus looked for in their offspring. The expression of said virus genes (if there is any) could be followed in rodent tissues.

Borna disease PCR without reverse-transcriptase. A) no nuclease treated, B) RNA nuclease treated, C) DNA nuclease treated and D) PCR with reverse transcriptase step

Well one paper has maybe taken the first step in the development of such a model system (although they may not know it). It has shown evidence that if you infect baby bank voles with borna virus, directly into their brain you can detect borna virus-specific DNA sequences using PCR following DNA extraction (see above PCR gel for results).  And, these sequences resulted from the virus, not some already-endogenised borna virus sequence. Although they did not check for germ-line infection or integration, this is the first step. The applicability of Borna virus reverse genetics and these animal models could make this kind of study feasible but certainly not easy. We may in future catch a glimpse of this process in real-time.

Kinnunen, P., Inkeroinen, H., Ilander, M., Kallio, E., Heikkilä, H., Koskela, E., Mappes, T., Palva, A., Vaheri, A., Kipar, A., & Vapalahti, O. (2011). Intracerebral Borna Disease Virus Infection of Bank Voles Leading to Peripheral Spread and Reverse Transcription of Viral RNA PLoS ONE, 6 (8) DOI: 10.1371/journal.pone.0023622