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 180: Throwing IFIT at flu and holding a miR to HCV

On episode #180 of the science show This Week in Virology, Vincent, Alan, and Rich review association of an interferon-induced protein with severe influenza, and stabilization of HCV RNA by a microRNA.

You can find TWiV #180 at www.microbe.tv/twiv.

TWiV 97: California virology

Hosts: Vincent Racaniello, Peter Sarnow, and Bert Semler

On episode #97 of the podcast This Week in Virology, Vincent visited Peter Sarnow and Bert Semler during a trip to California, and spoke with them about their work on internal ribosome entry, and the requirement for a cellular microRNA for hepatitis C virus replication.

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Click the arrow above to play, or right-click to download TWiV #97 (66 MB .mp3, 91 minutes)

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with Stitcher Radio.

Links for this episode:

  • Eukaryotic mRNAs that might contain an IRES (PNAS)
  • Modulation of HCV RNA abundance by a liver-specific microRNA (Science)
  • Viral small RNAs (PLoS Pathogens)
  • Bridging IRES elements to the translation apparatus (Biochim Biophys Acta)
  • A nucleo-cytoplasmic SR protein functions in viral IRES mediated translation (EMBO J)
  • Nuclear vs cytoplasmic routes to IRES mediated translation (Trends in Microbiology)
  • Letter read on TWiV 97

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv or leave voicemail at Skype: twivpodcast. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

Hantavirus protein replaces eIF4F

eif4fThe cellular translation machinery is frequently modified in virus-infected cells. Antiviral defense systems or stress responses may be initiated to inhibit protein synthesis and restrict virus replication. On the other hand, many viral genomes encode proteins that modify the cellular translation apparatus to favor the production of viral proteins over those of the cell. One such well-studied modification is the cleavage of the cellular translation protein eIF4G (see illustration) in cells infected by picornaviruses. The consequence of this modification is that capped cellular mRNAs cannot be translated. As the viral genomes are translated by internal ribosome entry, viral protein synthesis is not affected by cleavage of eIF4G.

A recent report in The EMBO Journal has revealed a novel modification of the cellular translation apparatus in cells infected with Sin Nombre virus, a hantavirus.The authors show that the viral nucleocapsid (N) protein binds with high affinity to the cap structure on cellular mRNAs. The N protein can also bind the 43S preinitiation complex (which consists of the 40S ribosomal subunit, several initiation proteins, and the met-tRNAi). Finally, N protein has RNA helicase activity, which facilitates ribosome movement through areas of RNA secondary structure. This viral protein therefore functionally replaces  all three components of eIF4F: eIF4E (the cap-binding protein), eIF4G (the scaffolding protein which connects the ribosome to the mRNA), and eIF4A, an RNA helicase. It does so even though it has no amino acid similarity to the proteins of eIF4F. Furthermore, the N protein was previously shown to be involved in viral RNA replication and encapsidation. The multifunctional nature of the N protein should come as no surprise: the hantavirus genome encodes only four proteins. Each must therefore fulfill multiple functions in the replication cycle.

Why would the hantavirus genome encode a protein that replaces eIF4F? One of the earliest cellular responses to virus infection is inhibition of translation;the goal is to restrict viral spread. The properties of the N protein could enable unabated viral translation in the face of such a cellular defense. Furthermore, many viral genomes encode proteins that inhibit viral translation. No such activity has been described in cells infected with hantaviruses. Nevertheless, the N protein could permit translation of viral mRNAs when that of cellular mRNAs is inhibited.

The participation of the hantavirus N proteins in multiple events in the cell identify it as an excellent target for therapeutic intervention.

Mohammad A Mir, Antonito T Panganiban (2008). A protein that replaces the entire cellular eIF4F complex The EMBO Journal, 27 (23), 3129-3139 DOI: 10.1038/emboj.2008.228

Poliovirus is IRESistable

Our latest paper has just been published in the Journal of Clinical Investigation. The title of the paper is “Poliovirus tropism and attenuation are determined after internal ribosome entry”. This is the work of a Ph.D. student in my laboratory, Steven Kauder.

If you would like a nice summary of this work, there is an excellent commentary by Bert Semler in the same journal, entitled “Poliovirus proves IRES-istible in vivo“. The title of this commentary is a play on the main theme of the research paper: the Internal Ribosome Entry Site (IRES) of poliovirus. The poliovirus IRES is an RNA sequence at the 5′-end of the viral genome that allows ribosomes to bind internally, rather than threading on the 5′-end as they do for most mRNAs. In our paper, we show that poliovirus attenuation and tropism are not determined by the viral IRES.

Let’s back up a bit to explain this last statement. Viral tropism is defined as the tissues in which a virus replicates. Poliovirus, the causative agent of poliomyelitis, infects very few tissues in humans: the intestine, the brain and spinal cord, and perhaps one other site. A restricted tropism is in fact a common property of many viruses. What restricts viral multiplication to so few tissues has been a long-standing question in virology. For poliovirus, it was first believed that the restricted tropism was a consequence of where the virus receptor is located. The virus receptor is a cell surface protein that is needed to bind the virus particle and bring the genetic material of the virus into the cell. However, some time ago it was shown that the receptor for poliovirus does not determine the narrow tropism of the virus. Subsequently it was suggested that the viral IRES might control the tropism – but in this recent paper we show that this is not the case.

The other topic of our paper concerns the live poliovirus vaccine, also known as the Sabin vaccine or oral poliovirus vaccine (OPV). There are three different vaccine strains of poliovirus, all isolated by Albert Sabin. The genetic material of each vaccine strain contains mutations, or genetic changes, that prevent it from causing disease. When the Sabin vaccines are ingested, they replicate in the intestine and provide immunity to infection, but they do not cause polio. Precisely how these mutations ‘work’ has been a matter of considerable debate. It has been believed that the mutations change the properties of the viral IRES so that it continues to direct translation in the human gut, but not in the spinal cord and brain. In our paper we show that this hypothesis is wrong. A mutation in one of the three Sabin vaccine strains actually weakens the virus in all tissues.

I recognize that much of this description may be beyond the understanding of someone who is not a scientist. A goal of this weblog is to make virology accessible to everyone. Therefore in the coming weeks I will endeavor to provide the background needed to understand this and similar material that will appear here.