Priming RNA synthesisThe enzymes that make copies of the DNA or RNA genomes of viruses – nucleic acid polymerases – can be placed into two broad categories depending on whether or not they require a primer, a short piece of DNA or RNA, to get going. The structure of the primer-independent RNA polymerase of hepatitis C virus reveals how a priming platform is built into the enzyme.

The requirement for a primer in the initiation step of nucleic acid synthesis varies among the different classes of polymerases. All DNA polymerases are primer-dependent enzymes, while DNA-dependent RNA polymerases initiate RNA synthesis de novo – without a primer. Some RNA-dependent RNA polymerases can also initiate RNA synthesis without a primer: the enzyme begins by adding the first base complementary to the template RNA (illustrated). Other RNA-dependent RNA polymerases require a primer to initiate synthesis. Examples shown on the illustration include the protein-linked primer of picornaviruses, which consists of the protein VPg covalently attached to two U residues. The primer for influenza virus mRNA synthesis is a capped oligonucleotide 12-14 bases in length that is cleaved from the 5′ end of cellular mRNA.

The structure of the RNA-dependent RNA polymerase of hepatitis C virus reveals how a primer-independent RNA polymerase positions the first nucleotide on the RNA template. This process is illustrated below. With the RNA template (dark green) in the active site of the enzyme (panel A), a short beta-loop (brown) provides a platform on which the first complementary nucleotide (light green) is added to the template. The second nucleotide is then added (panel B), producing a dinucleotide primer for RNA synthesis. At this point nothing further can happen because  the priming platform blocks the exit of the RNA product from the enzyme (panel B). The solution to this problem is that the polymerase undergoes a conformational change that moves the priming platform out of the way and allows the newly synthesized complementary RNA (panel C, light green) to exit as the enzyme moves along the template strand.

 HCV priming of RNA synthesis

The structure of the RNA polymerase of hepatitis C virus reveals that it is not really a primer-independent enzyme: a dinucleotide primer is synthesized by the polymerase using a protein platform in the active site. Such protein platforms also appear to be involved in the priming of RNA synthesis by other flaviviruses (dengue and West Nile viruses), influenza virus (genome RNA synthesis is primer independent), reovirus, and bacteriophage phi6. Perhaps all viral RNA-dependent RNA polymerases are dependent on such priming platforms to initiate RNA synthesis.

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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.twiv.tv.

Sabin type 2 poliovirusViruses can be broadly classified according to whether or not the particle is enveloped – surrounded by a membrane taken from the host cell – or naked. Some naked viruses apparently are more modest than we believed.

Members of the family Picornaviridae, which include Hepatitis A virus, poliovirus, and Coxsackieviruses, have non-enveloped particles that consist of a protein shell surrounding the viral RNA genome (poliovirus is illustrated). Examples of viruses that are enveloped include dengue virus, influenza virus, and measles virus.

Recently it was discovered that hepatitis A virus (HAV) particles are released from cells in membrane vesicles containing 1-4 virus particles. These membranous structures resemble exosomes, which are also released from uninfected cells and play roles in various biological processes. Enveloped hepatitis A virus particles are present in the blood of infected humans. However virus in the feces, which is transmitted to other hosts, is not enveloped.

Viral envelopes typically contain viral glycoproteins, such as the HA protein of influenza viruses, which serve important functions during replication, such as attachment to cell receptors. Envelope glycoproteins are also the target of antibodies that block viral infection. The presence of an envelope makes HAV resistant to neutralization with antibodies, because the membrane contains no viral proteins that can be blocked by antibodies.

Two other non-enveloped picornaviruses, Coxsackievirus B and poliovirus, are also released from cells within membrane vesicles. These virus particles are in vesicles derived from the autophagy pathway, which captures and recycles cytoplasmic contents by ejecting them from the cell.

What is the function of the membrane acquired by these naked viruses? Perhaps immune evasion: the presence of the cell membrane makes HAV and Coxsackievirus B virus particles resistant to neutralization with antibody. The ability to deliver multiple virus particles to a single cell might help to overcome genetic defects in the viral genome that are a consequence of the high mutation rates of these viruses.

An interesting problem is how these cloaked viruses enter cells, because there is no evidence that the membranes contain any viral proteins that could interact with a cell receptor. Nevertheless, entry of enveloped HAV and poliovirus into cells requires the known viral receptor. Perhaps the vesicles are taken into the cell by endocytosis, where viral particles are released from the vesicles, and then bind receptors to initiate escape of the genome.

Should HAV, poliovirus, and Coxsackievirus B be reclassified as enveloped viruses? Probably not, in part because the membranes surrounding these virus particles are not needed for infectivity. In contrast, removal of the membrane from influenza virus, dengue virus, or measles virus destroys their infectivity. Enveloped viruses acquire a membrane after the internal components have been assembled, whether they are helical or icosahedral nucleocapsids. In contrast, HAV, poliovirus, and Coxsackievirus B become fully infectious particles before they acquire an envelope.

Another argument against calling picornaviruses enveloped is that viral membranes contain viral glycoproteins that allow attachment to cell receptors and release of the viral genome into the cell. There is no evidence that the membranes of picornaviruses contain viral proteins.

The acquisition of a membrane may have taken place later in the evolution of picornaviruses, to allow more efficient infection or evasion of host responses. Alternatively, the membrane may simply be a by-product acquired when these viruses exit the cell by a non-lytic mechanism.

While the finding of membranes around picornavirus particles is intriguing, I am not yet convinced that these viruses should be considered to be enveloped. I would like to know if other non-enveloped viruses are similarly released from cells in membranous cloaks, and the function of this addition for viral replication in the host.

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On episode #328 of the science show This Week in Virology, the TWiVocateurs discuss how the RNA polymerase of enteroviruses binds a component of the splicing machinery and inhibits mRNA processing.

You can find TWiV #328 at www.twiv.tv.

Areas with CWDChronic wasting disease (CWD) is a prion disease of cervids (deer, elk, moose). It was first detected in Wyoming and Colorado, and has since spread rapidly throughout North America (illustrated; image credit). Because prions that cause bovine spongiform encephalopathy (BSE, mad cow disease) are known to infect humans, there is concern that CWD might also cross the species barrier and cause a novel spongiform encephalopathy. Recent experimental results suggest that CWD prions are not likely to directly infect humans.

The prion protein PrPC is encoded by the prnp gene, which is essential for the pathogenesis of transmissible spongiform encephalopathies (TSEs). Transgenic mice have been used to understand the species barrier to prion transmission. When mice are inoculated with human prions, few animals develop disease and the incubation periods are over 500 days. When the mouse prnp gene is replaced with the human gene, the mice become uniformly susceptible to infection with human prions and the incubation period is shorter. The species barrier to prion transmission is therefore associated with differences in the prion protein sequence between host and target species.

Mice have been used to understand whether CWD prions might be transmitted to humans. Mice are not efficiently infected with CWD prions unless they are made transgenic for the cervid prnp gene. Four different research groups have found that  mice transgenic for the human prnp gene are not infected by CWD prions. These findings suggest that CWD prions are not likely to be transmitted directly to humans. However, changing four amino acids in human prnp to the cervid sequence allows efficient infection of transgenic mice with cervid prions.

Another concern is that prions of chronic wasting disease could be transmitted to cows grazing in pastures contaminated by cervids. Prions can be detected in deer saliva and feces, and contamination of grass could pass the agent on to cows. In the laboratory, brain homogenates from infected deer can transmit the disease to cows. Therefore it is possible that cervid prions could enter the human food chain through cows.

A further worry is that BSE prions shed by cows in pastures might infect cervids, which would then become a reservoir of the agent. BSE prions do not infect mice that are transgenic for the cervid prnp gene. However, intracerebral inoculation of deer with BSE prions causes neurological disease,  and the prions from these animals can infect mice that are transgenic for the cervid prnp gene. Therefore caution must be used when using transgenic mice to predict the abilities of prions to cross species barriers.

Although the risk of human infection with CWD prions appears to be low, hunters should not shoot or consume an elk or deer that is acting abnormally or appears to be sick, to avoid the brain and spinal cord when field dressing game, and not to consume brain, spinal cord, eyes, spleen, or lymph nodes. No case of transmission of chronic wasting disease prions to deer hunters has yet been reported.

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On episode #327 of the science show This Week in Virology, the eTWiVicators review evidence that the HIV-1 group O epidemic began with a single cross-species transmission of virus from western lowland gorillas.

You can find TWiV #327 at www.twiv.tv.

poliovirus + receptorWe recently discussed the development of a soluble receptor for HIV-1 that provides broad and effective protection against infection of cells and of nonhuman primates. Twenty-five years ago my laboratory published a paper which concluded that using soluble receptors to block virus infection might not be a good idea. In the first paragraph of that paper we wrote:

…it has been proposed that soluble cell receptors might be effective antiviral therapeutics. It has been suggested that mutants resistant to the antiviral effects of soluble receptors would not arise, because mutations that abrogate binding to receptors would be lethal.

We had previously shown that the cell receptor for poliovirus, CD155, produced in a soluble form, would bind to poliovirus (pictured – the very image from the banner of this blog), blocking viral infection. We then found that it was relatively easy to select for soluble receptor resistant (srr) virus mutants. These viruses still enter cells by binding to CD155, but the affinity of virus for the receptor is reduced. Poliovirus srr mutants replicate normally in cell cultures, and cause paralysis in a mouse model for poliomyelitis. We speculated that receptor binding might not be a rate-limiting step in viral infection, and short of  abolishing binding, the virus can tolerate a wide range of binding capabilities.

The amino acid changes that cause the srr phenotype map to both the exterior and the interior of the viral capsid. The changes on the virion surface are likely to directly interact with the cell receptor. Changes in the interior of the virus particle may be involved in receptor-mediated conformational transitions that are believed to be essential steps in viral entry.

When this work was done, clinical trials of soluble CD4 for HIV-1 infection were under way. We believed that our findings did not support the use of soluble receptors as antivirals, which we clearly stated in the last sentence of the paper:

These findings temper the use of soluble receptors as antiviral compounds.

HIV-1 mutants resistant to neutralization with soluble CD4 were subsequently isolated, and the compound was never approved to treat HIV-1 infection in humans for this and other reasons, including low affinity for the viral glycoprotein, enhancement of infection, and problems associated with using a protein as a therapeutic.

Recently a new soluble CD4 was produced which also includes the viral binding site for a second cell receptor, CCR5. This molecule overcomes many of the issues inherent in the original soluble CD4. It provides broad protection against a wide range of HIV-1 strains, and when delivered via an adenovirus-associated virus vector, protects nonhuman primates from infection. This delivery method circumvents the issues inherent in using a protein as an antiviral drug. Because this protein blocks both receptor binding sites on the viral envelope glycoprotein, it might be more difficult for viruses to emerge that are resistant to neutralization. The authors speculate that such mutants might not be efficiently transmitted among hosts due to defects in cell entry. Given the promising results with this antiviral compound, experiments to test this speculation are certainly welcome.

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On episode #326 of the science show This Week in Virology, the sternutating TWiVers discuss preventing infection of cells and animals by a soluble CD4-CCR5 molecule that binds to HIV-1 virus particles.

You can find TWiV #326 at www.twiv.tv.

eCD4-FcBecause viruses must bind to cell surface molecules to initiate replication, the use of soluble receptors to block virus infection has long been an attractive therapeutic option. Soluble receptors have been developed that block infection with rhinoviruses and HIV-1, but these have not been licensed due to their suboptimal potency. A newly designed soluble receptor for HIV-1 overcomes this problem and provides broad and effective protection against infection of cells and of nonhuman primates.

Infection with HIV-1 requires two cell surface molecules, CD4 and a chemokine receptor (either CCR5 or CXCR4), which are engaged by the viral glycoprotein gp120 (illustrated). A soluble form of CD4 fused to an antibody molecule can block infection of most viral isolates, and has been shown to be safe in humans, but its affinity for gp120 is low. Similarly, peptide mimics of the CCR5 co-receptor have been shown to block infection, but their affinity for gp120 is also low.

Combining the two gp120-binding molecules solved the problem of low affinity, and in addition provided protection against a wide range of virus isolates. The entry inhibitor, called eCD4-Ig, is a fusion of the first two domains of CD4 to the Fc domain of an antibody molecule, with the CCR5-mimicking peptide at the carboxy-terminus (illustrated). It binds strongly to gp120, and blocks infection with many different isolates of HIV-1, HIV-2, SIV, and HIV-1 resistant to broadly neutralizing monoclonal antibodies. The molecule blocks viral infection at concentrations that might be achieved in humans (1.5 – 5.2 micrograms per milliliter).

When administered to mice, eCD4-Ig protected the animals from HIV-1. Rhesus macaques inoculated with an adenovirus-associated virus (AAV) recombinant containing the gene for eCD4-Ig were protected from infection with large amounts of virus for up to 34 weeks after immunization. Levels of eCD4-Ig in the sera of these animals ranged from 17 – 77 micrograms per milliliter.

These results show that eCD4-Ig blocks HIV infection with a wide range of isolates more effectively than previously studied broadly neutralizing antibodies. Emergence of HIV variants resistant to neutralization with eCD4-Ig would likely produce viruses that infect cells less efficiently, reducing their transmission. eCD4-Ig is therefore an attractive candidate for therapy of HIV-1 infections. Whether sustained production of the protein in humans will cause disease remains to be determined. Because expression of the AAV genome persists for long periods, it might be advantageous to include a kill-switch in the vector: a way of turning it off if something should go wrong.

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TWiV 325: Wildcats go viral

22 February 2015

On episode #325 of the science show This Week in Virology, Vincent visits the ‘Little Apple’ and speaks with Rollie and Lorena about their work on mosquito-born viruses and baculoviruses.

You can find TWiV #325 at www.twiv.tv.