Hot plants and viruses, part III: An inordinate fondness for hypothetical proteins

Grasses at Yellowstone National Park are able to grow temperatures (65°; C) that would toast most living things.

Step right up! Watch original research, as it happens, on the web!

I'm going to use bioinformatics to see if I can find that answer to the puzzle of heat-tolerant plants.

Previous parts:

• Part I. The research
• Part II. An introduction

Today: Part III: An inordinate fondness for hypothetical proteins

After today, we will have:

• Part IV: Did we get lost in translation?

(I will add the link after it's posted)

Let's begin

Sigh.

It always happens when I have homework to grade. An intriguing story comes along and before I know it I've got 20 web browser windows open, I've embarked on a bioinformatics research project, and I'm reading abstracts trying to solve the mystery.

It's irresistible.

I know it's kind of strange and geeky, but I've been really intrigued by the question of a why a virus-infected fungus helps plants grow at hot temperatures. So I decided to find out and as you'll see later in this series, I think I did. I'm going to present the results of this research in a few more parts, so I can walk through each part and show where the authors of the paper made a mistake.

All about the virus
In the paper by Márquez, et. al. (1), the authors noted the following characteristics of the double-stranded RNA virus:

Sequence analysis revealed that each of the two RNA segments contains two open reading frames (ORFs) (fig. S2). The 2.2-kb fragment (RNA 1) is involved in virus replication, as both of its ORFs are similar to viral replicases. The first, ORF1a, has 29% amino acid sequence identity with a putative RNA-dependent RNA polymerase (RdRp) from the rabbit hemorrhagic disease virus. The amino acid sequence of the second, ORF1b, has 33% identity with the RdRp of a virus of the fungal pathogen Discula destructiva. These two ORFs overlap and could be expressed as a single protein by frameshifting, a common expression strategy of viral replicases. The two ORFs of RNA 2 have no similarity to any protein with known function. As in most dsRNA mycoviruses, the 5' ends (21 bp) of both RNAs are conserved.

I was intrigued by RNA 2 and the identity of the two unidentified open reading frames. What could they be?

Like many papers that include sequence analysis, the authors didn't give any information about the searches they did or why they arrived at this conclusion. So, I thought I'd begin by trying to reproduce their results with RNA 2.

I got the nucleotide sequence accession numbers from their paper and used blastp to search the nr (non-redundant) protein database at the NCBI with each of the predicted protein sequences.

Search results from the first ORF. Click this image to see a larger version.

Does Aspergillus harbor double-stranded RNA viruses?
In the first set of search results, I found significant matches (E values of < 2 x 10^-14 and below). But these were to hypothetical proteins. Since all the matches are to hypothetical proteins, this result isn't too exciting.

The sequences were hypothetical proteins from another fungus, Aspergillus, though, which is interesting. I looked at the GenBank records and found that at least one of those sequences corresponded to an RNA transcript from Aspergillus. This makes me wonder if that strain of Aspergillus was infected by an RNA virus.

Results from a blastp search with the second ORF

The second protein didn't give me any significant matches.

This probably why the researchers stopped searching and let the story end there.

Not me, though. Not only do I have homework to grade, I know other ways to search.

Where's the rest of the story? Continue and see.

Reference:
Márquez, L., et. al. 2007 A Virus in a Fungus in a Plant: Three-Way Symbiosis Required for Thermal Tolerance Science 26: 513-515.