A virus with a green thumb

PoxvirusI just love it when long standing mysteries in virology are suddenly solved, typically by the use of new technologies. In this story, the long standing mystery was why poxvirus mRNAs have a stretch of poly(A) in their 5′-noncoding regions. The answer is that it allows the ribosome to preferentially translate these viral mRNAs over those of the host (link to paper).

Ribosomes, the sites of protein synthesis, are very large assemblies of RNAs and proteins. A ribosome-associated protein called RACK1 is the major player in this story. This protein was previously found to be important for translation of viral RNAs by internal ribosome entry, a process in which ribosomes bypass the 5′-cap structure on mRNAs.

Cells lacking the gene encoding RACK1 seem to be just fine – they grow normally. This result would suggest that RACK1 is not needed for translation of mRNAs via the 5′-cap structure. But the translation of mRNAs of vaccinia virus, a poxvirus, is blocked in cells lacking RACK1. More specifically, the viral mRNAs produced later in infection cannot be translated.

This effect of RACK1 on vaccinia virus mRNA translation prompted a closer look at the protein in virus infected cells – which revealed that it is phosphorylated by a viral protein kinase. Phosphorylation of RACK1 makes the ribosomes preferentially translate vaccina virus mRNAs – an effect that is completely dependent upon the poly(A) in the 5′-untranslated region! Transfer of this poly(A) sequence to a non-viral mRNA enhanced its translation in cells in which RACK1 is phosphorylated.

The amino acid sequence of RACK1 that is phosphorylated is within a loop that contacts the 18S ribosomal RNA subunit. An examination of RACK1 proteins from different species reveals that in plants, unlike in mammals, this loop contains a stretch of negatively charged amino acids. Because a phosphate is negatively charged, it made perfect sense to see if the plant loop sequence, when transferred to mammalian RACK1, could stimulate translation of poly(A) containing mRNAs. It did!

In other words, phosphorylation by a vaccinia virus protein kinase makes RACK1 plant-like! Which is why the authors called their paper ‘Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase”. Exactly what RACK1 protein is doing in plants is a burning question, as is how RACK1 recognizes mRNAs containing a poly(A) leader.

Phosphorylation of RACK1 by the vaccinia virus kinase explains why viral mRNAs with poly(A) leader sequences are preferentially translated in infected cells (late viral mRNAs have this unusual leader). The presence of poly(A) in the 5′-ends of late vaccinia virus mRNAs is considered to be a consequence of polymerase slippage – but it is clearly not a mistake. At one time, long ago, a polymerase might have slipped for the first time, but the resulting poly(A) in the mRNA conveyed a selective advantage that remained. Today I would no longer call the addition of poly(A) to vaccinia virus mRNAs an error!

Why did it take so long to figure out the role of poly(A) at the 5’-end of vaccinia virus mRNAs? The right technology was not available – the key ones were the ability to knock out specific genes in cells, to knock down mRNA levels with RNA interference, and to identify the sites of RACK1 phosphorylation by mass spectrometry. These are not only recently developed techniques, but they have become widely accessible. But don’t forget the use of long-ago isolated vaccinia virus mutants of the protein kinase that were an essential part of this story.

Rich Condit, a poxvirologist, joined Nels Elde (also a poxvirologist, though he dabbles in other systems) and me to discuss this very cool paper on episode #21 of TWiEVO, This Week in Evolution. Rich had some very good insight on this paper; plus he bristled twice during the episode. You’ll have to listen to find out why.

Image credit.

 

TWiV 351: The dengue code

On episode #351 of the science show This Week in Virology, the Masters of the ScienTWIVic Universe discuss a novel poxvirus isolate from an immunosuppressed patient, H1N1 and the gain-of-function debate, and attenuation of dengue virus by recoding the genome.

You can find TWiV #351 at www.microbe.tv/twiv.

TWiV 329: Pox in the balance

On episode #329 of the science show This Week in Virology, the TWiV team reviews identification of immune biomarkers in CFS/ME patients, and how a cell nuclease controls the innate immune response to vaccinia virus infection.

You can find TWiV #329 at www.microbe.tv/twiv.

TWiV 244: Back in the CVVR

Episode #244 of the science show This Week in Virology was recorded before an audience at the Beth Israel Deaconess Medical Center, where Vincent and Alan spoke with Dan and Jeff about AIDS vaccines.

You can find TWiV #244 at www.microbe.tv/twiv.

TWiV 235: Live in Edmonton, eh?

Episode #235 of the science show This Week in Virology was recorded before an audience at the 2nd Li Ka Shing Institute of Virology Symposium at the University of Alberta, where they spoke with Dave, Stan, and Lorne about their work on poxvirus vaccines and recombination, an enveloped picornavirus, antivirals against hepatitis B and C viruses, and supporting virology research in Alberta.

You can find TWiV #235 at www.microbe.tv/twiv.

TWiV 234: Live in Denver

Episode #234 of the science show This Week in Virology was recorded before an audience at the 2013 General Meeting of the American Society for Microbiology in Denver, Colorado. Vincent and Kathy spoke with Nels Elde and Tom Shenk about their work on the evolution of virus-host conflict and how viruses influence the cell metabolome.

You can find TWiV #234 at www.microbe.tv/twiv.

TWiV 213: Not bad for a hobby

On the final episode of the year of the science show This Week in Virology, the TWiV team reviews twelve cool virology stories from 2012.

You can find TWiV #213 at www.microbe.tv/twiv.

TWiV 203: Mark Challberg, a cold room kind of guy

On episode #203 of the science show This Week in Virology, Vincent and Rich meet up with Mark Challberg to talk about his scientific career studying viral DNA replication, and his transition to an NIH Program Officer.

You can find TWiV #203 at www.microbe.tv/twiv.

TWiV 198: Pox has got a squeeze-box, seals are gonna sneeze all night

On episode #198 of the science show This Week in Virology, Vincent, Alan, Rich, and Kathy review fatal avian influenza virus in harbor seals, and poxvirus deployment of genomic accordions to counter antiviral defenses.

There once was a virus named pox
Whose genome contained a squeeze-box
When placed under pressure
It expanded its measure
Overcoming the new cellular blocks

You can find TWiV #198 at www.microbe.tv/twiv.

TWiV 181: ORFan poxviruses and nIRFing prions

On episode #181 of the science show This Week in Virology, Vincent, Rich, and Kathy discuss Cotia virus, a new poxvirus, Orf virus infections associated with handling goats and lamb, and the innate immune response to prions.

You can find TWiV #181 at www.microbe.tv/twiv.