Tuesday, November 4, 2014

Stuttering Increases Filovirus Vocabulary

In a paper published by Shabman et. al. in mBio, the open-access journal of the American Society for Microbiology, today entitled "Deep Sequencing Identifies Noncanonical Editing of Ebola and Marburg Virus RNAs in Infected Cells" researchers identified a novel mechanism by which Filoviruses expand their mRNA repertoire.  The two notorious Filoviruses examined in this study were Ebola (specifically the same Zaire strain currently present in West Africa) and Marburg (specifically the Angola strain last seen in a 2005 outbreak in the country for which it is named).  Filoviruses have a negative-sense single-stranded genome that encodes several different genes with unique 'start' and 'stop' indicators for transcription.  It was traditionally thought that each gene produced a single mRNA and therefore a single protein, except for the Ebola GP gene.  The Ebola GP gene was known to produce three possible glycoproteins (GP, sGP and ssGP) through a process called RNA editing in which the viral polymerase adds 'A' nucleotides that are not encoded by the genome itself.

This research team leveraged the power of next-generation sequencing, which can offer high-resolution  data on nucleotide sequences, to examine mRNAs, the products of genomic transcription, and reveal new insights into the regulation of filovirus gene expression.  Their results revealed a 3′-to-5′ transcriptional gradient previously documented in other negative-sense single-stranded viruses. This gradient means that genes closer to the 3' end are transcribed more than those on the 5' end.  The viral polymerase was found to obey the 'start' and 'stop' transcription signals fairly strictly, with limited 'read-through' and therefore limited transcription of intergenic regions.

Beyond these common regulatory features, the study also revealed novel gene-product diversification mechanisms.  'Hot spots' in the Ebola GP gene mRNA and Marburg NP 3′-UTR gene mRMA were shown to be modified by host cytosine deaminase enzymes (generating U->C substitutions).  While this editing was relatively infrequent, 25-30% of the GP mRNA showed evidence of non-templated 'A' nucleotides added through viral polymerase 'stuttering.'  Beyond the GP region previously shown to undergo this editing, the team found two other sites of nucleotide addition at similar homopolymer sites of  6-A or 6-U.  Nucleotide addition was also demonstrated, for the first time, at sites in Marburg mRNAs, specifically single-nucleotide insertions in NP and L mRNAs.  Interestingly, this non-cannonical editing was lower in infected animal models than in tissue culture and was highly conserved at particular mRNA sites.

Overall, this study represents an important step forward in scientific understanding of deadly filoviruses.  These authors revealed that the virus family has greater complexity and diversity of gene expression than previously thought, which indicates both a need for further research to reveal the functional implications of this editing and more innovative strategies to combat filovirus infection.

Read the full paper here:  http://mbio.asm.org/content/5/6/e02011-14.full

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