Genes must be expressed in precise levels and at the exact moment if the complex balance regulating cell activity is to be maintained. Messenger RNA (mRNA) conveys genetic information from DNA to ribosomes, where proteins are synthesized through the linkage of amino acids. These amino acids are supplied by fragments of transfer RNA (tRNA), which decode the information contained within the mRNA codons (group of three RNA bases specifying each amino acid) in order to stitch them in the correct sequence to synthesize each protein. As such, translation of mRNAs into proteins forms a crucial step during gene expression.
Under stressful conditions, cells regulate their gene expression in several ways; translation initiation has been reported to be impacted during cellular stress. Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA, usually an AUG. Viral infection is a stressful event for a cell, and has been shown to affect translation initiation, particularly to alter the start codon on mRNAs at which ribosomes initiate translation. A common alternate start codon, CUG, may be preferred in times of immune stress. Furthermore, alternate translation initiation is one method viruses often utilize to increase the diversity of proteins encoded in their compact genomes.
Ribosome profiling, a genome-wide method to study translation by deep sequencing ribosome-protected mRNA fragments, has been used to examine changes in translation on host transcripts during influenza virus infection. However, these studies did not include the use of inhibitors of translation initiation; there is a critical need to comprehensively identify sites of translation initiation during influenza virus infection. Basic Sciences Division faculty members Drs Rasi Subramaniam and Jesse Bloom teamed up, together with their graduate student Heather Machkovech, to do just that. In their paper published in a recent issue of PLOS Pathogens, the authors integrated computational and experimental approaches to systematically investigate the extent and impact of alternate translation in cells infected with influenza virus.
The authors first performed computational analyses that suggest there is selection against putative CUG alternate translation initiation sites in mammalian influenza virus lineages. They then used ribosome profiling to experimentally map sites of translation initiation in viral and cellular mRNAs during influenza virus infection. Surprisingly, there was little signal for CUG initiation in viral genes, but a number of candidate AUG start sites downstream of the canonical start codon were identified. They demonstrated that one of these candidate start sites leads to production of a truncated version of the viral neuraminidase protein expressed on the cell surface and is enzymatically active.
On the host side, the authors identify an excess of non-canonical start codons on transcripts that are upregulated during the anti-viral response. Together, this work highlights how alternate translation initiation can alter the protein-coding capacity of both viruses and the cells that they infect.
Dr. Rasi Subramaniam on the novelty of this study: “Our comprehensive profiling of translation initiation represents an unbiased strategy for identifying alternate protein products during viral infections. Our work adds to a growing body of evidence that, despite their small genome size, viruses greatly expand the diversity of the proteins that they produce by alternate translation mechanisms.”
Dr. Jesse Bloom explains the significance of this work: "This is the first study to comprehensively map the sites of protein translation initiation in influenza-virus infected cells. There are two reasons this is important: it can identify new peptides produced by the virus, and it can identify changes in translation that increase production of immune epitopes. We found a new peptide produced by the virus, although we were unable to show that it had functional significance. Perhaps more surprisingly, although we found a lot of circumstantial evidence suggesting that changes in translation can produce epitopes, we could not identify direct evidence for this."
Both host and viral mRNAs are translated into proteins when viruses such as influenza infect cells. This study demonstrates that alternate translation initiation plays a role in shaping the repertoires of both viral and host proteins that are produced during influenza infection, and sheds light on the evolutionary forces shaping this landscape. Dr. Subramaniam reveals what lies ahead: “It will be exciting to identify mechanisms by which hosts suppress this viral diversity or use it for enhancing their immune response. Our labs are currently studying whether host cells also use alternate translation as a mechanism to respond to viral infections.”
Machkovech HM, Bloom JD, Subramaniam AR. 2019. Comprehensive profiling of translation initiation in influenza virus infected cells. PLoS Pathog, 15(1), e1007518.
Funding was provided by the National Institutes of Health and the Howard Hughes Medical Institute.
Basic Sciences Division
Human Biology Division
Maggie Burhans, Ph.D.
Public Health Sciences Division
Vaccine and Infectious Disease Division
Clinical Research Division
Julian Simon, Ph.D.
Clinical Research Division
and Human Biology Division
Arnold Digital Library