Controversial Influenza Research

REFERENCE: Imai et al. "Experimental Adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets." Nature (2012), epub ahead of print.  LINK

Amedeo Post offering more background and editorials related to this work: LINK

                This week in Nature sees the publication of the long discussed influenza research paper from the University of Wisconsin-Madison lab of Yoshihiro Kawaoka.  In a recent editorial, Kawaoka urges for publication and further research in this area by saying “[s]ome people have argued that the risks of such studies…outweigh the benefits.  I counter that H5N1 viruses circulating in nature already pose a threat because influenza viruses mutate constantly and can cause pandemics with great losses of life.  …I believe it would irresponsible not to study the underlying mechanisms.”  This published paper focuses on the hemaglutinin (HA) protein from the H5N1 virus circulating primarily in Southeast Asia that retains specificity and virulence in birds.  To date, 578 humans have become infected with this virus after direct contact with infected animals.  340 have died, but human-to-human transmission has, so far, not been an issue, but the potential for a pandemic caused by an evolving H5N1 virus is still present.  Kawaoka and colleagues sought to determine what mutations within this H5 would allow the protein to bind human receptors, if an influenza virus bearing these mutations could both efficiently infect and transmit the virus among mammals, and finally if current vaccines/antiviral therapies would be useful against such a virus.

                Human cells of the respiratory tract display α2,6-linked sialic acid with galactose while avian tracts have α2,3 linkages.  As the main receptors for HA binding, the ability of an influenza virus that was specific for birds to infect humans means, in part, that the specificity for binding switched from α2,3 to α2,6.  The authors began by introducing random mutations into the H5 globular head region where receptor binding occurs.  Turkey red blood cells were treated with sialidase to remove α2,3 linked sialic acid and preferentially leave α2,6.  Viruses were generated bearing the mutated H5s and tested for their ability to bind treated turkey red blood cells.  The identified viruses were screened again for α2,6 binding to root out false-positives and identified HAs were then further tested in solid-phase binding experiments for α2,6-specificity.  In the end, an H5 bearing mutations at E119, V152, N224 and Q226 was identified as binding only α2,6 linkages.  The authors further confirmed that N224 and Q226 mutations were critical for the shift in specificity.

                The hemaglutinin from H1N1, which was isolated from a human patient, was replaced with an H5 bearing the appropriate N224 and Q226 mutations.  Ferrets were infected with this reassortant virus and found that, after a period of 6 days, a new mutation at position N158 was observed. Viruses bearing an H5 with this new additional mutation could replicate well in ferrets and were mildly transmissible between ferrets.  Intriguingly, the viruses were found to contain yet another mutation as position T318 after ferret infection.  A new H1N1 virus bearing a quadruple H5 mutant protein was found to be highly transmissible between ferrets.  No ferrets died as a result of infection with either virus.  Encouragingly, the authors also showed that a prototype H5N1 vaccine was reactive with the mutant viruses created here and the viruses were susceptible to a licensed NA inhibitor.

                Specific mutations at N224 and Q226 are mostly likely changing the binding pocket to accommodate α2,6 linkages; a specific N158 mutation removes a glycosylation site on H5 that could be improving transmissibility.  These three mutations destabilize the protein in an acidic environment but the T318 mutation returns that stability.  Membrane fusion of the influenza virus with the host cell occurs at low pH so stability at these [H⁺] is necessary.

                It should be noted that hemaglutinin, while highly involved, is only one protein involved in the virulence of an influenza virus.  Studies show that neuraminidase also plays a role.  It was also stressed that the remaining genes in the mutant virus came from H1N1, not the avian-virulent H5N1.  It’s possible that the remaining genes in the influenza also contribute to the virulence of a virus in new hosts.  The hope is that the amino acid mutations identified here will help those keeping an eye on current H5N1 viruses.  Should any of these mutations arise, they will have the tools to predict pandemic potential and, knowing that current therapies are effective against these viruses, that proper safeguards can be put in place quickly.  As a final thought, the authors realize that a pandemic virus may not even show these mutations and something else entirely, but the work has identified important areas of the HA protein that could and most likely will change as an influenza virus evolves from avian to mammal specificity.

 

Influenza Research Pause

REFERENCE: Fouchier et al. “Pause on avian flu transmission studies.”  Nature (2012)

LINK directly to published letter

                As I discussed in my American Society for Cell Biology Meeting post, I don’t want to repeat work that has already been discussed outside of the initial scientific publication, however this topic is interesting, especially considering the letter was signed by 39 authors and published in both Nature and Science magazines.  

                Work being performed at the University of Wisconsin-Madison and Erasmus MC in the Netherlands has suspended important research on a highly transmittable influenza virus due to fears of viral escape from their laboratories.  They have imposed a 60 day “pause” on their work while the scientific community and the community at large have time to discuss some of the new issues this type of research presents.

Ebolaviruses

REFERENCE: Dias et al. “A shared structural solution for neutralizing ebolaviruses.” (2011) Nature Structural and Molecular Biology 18(12) pgs 1424 – 1427.

                Five types of ebolaviruses have been identified: Sudan, Ebola, Reston, Bundibugyo, and Tai Forest.  The Sudan and Ebola forms cause the predominant amount of human deaths and recently a new variant of the Sudan virus has been found in the Gulu district of Uganda.  While many monoclonal antibodies exist, only a handful can neutralize Ebola virus and none can neutralize Sudan virus.  In a recent Nature Structural and Molecular Biology publication, Dias et al. discuss their development of a neutralizing antibody for Sudan virus and a subsequent crystal structure of it bound to the Gulu-Sudan variant protein GP_1,2.

                GP_1,2 (glycoprotein) is a viral trimeric receptor solely responsible for bringing ebolavirus into a host cell.  The protein is so named because the entire amino acid sequence is expressed then cleaved to create GP₁ and GP₂.  However, these two proteins remain attached to each other via a disulfide bond until the viral membrane fuses with the endosomal membrane.   

                The monoclonal antibody developed here, referred to as 16F6, recognized native Sudan virus GP_1,2 and their work indicated that binding of 16F6 alone was enough to block infection.  The epitope for the antibody was revealed by X-ray crystallography to be at the base of the trimer (Figure 11.1).  Further work showed that Sudan virus could still attach to host cells and be internalized, which left the authors to speculate that the antibody is either inhibiting another unidentified factor or it is blocking an additional necessary conformational change in GP_1,2 that leads to successful infection.  

                The final sentence of their paper summarizes a possible far-reaching conclusion from their work as “...for viruses in general, successful immunotherapy and vaccine design may depend on targeting antibodies that anchor glycoprotein subunits together and prevent the conformational changes required for fusion.”

Prions

Weissmann et al. “Prions on the move.” (2011) EMBO Reports 12(11) pgs 1109 – 1117.

             

                Prions are the infectious agents responsible for Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalopathy.  PrP^C is a 208 amino acid protein with two potential glycosylation sites.  Typically, it is found GPI-anchored to the plasma membrane outer surface.  PrP^SC is an aggregate of misfolded PrP molecules.  The aggregate recruits properly folded PrP to promote sequestration, protein misfolding and aggregate growth.

                A recent review by Browning and colleagues explores the recent literature to explain the leading theories on “barrier to transmission” and how prions can adapt to new environments.

                Consider a situation where the donor PrP is different in amino acid sequence from the recipient PrP.  Recipient PrP may have trouble joining the donor PrP aggregate for two reasons.  One, the differences in amino acid sequence may not allow the recipient PrP to adopt the necessary conformation needed for stable addition.  However, even when the PrP sequence is exactly the same, recipient PrP may still have problems, leading to the idea that different cellular environments and perhaps other proteins are involved in aggregate growth.

                An interesting study took 22L prions that could chronically infect PK1 cells in the presence of R33 cells and swainsonine.  Swainsonine is a small molecule that causes misglycosylation of proteins.  After forty population doublings, the prion population had become R33-incompetent and was sensitive to swainsonine.  When these new prions were placed back in the environment of the brain, the population changed back to being R33-competent and swainsonine insensitive.  

                The authors offer an excellent summation of these findings: “…a prion strain is a quasi-species, consisting of a major component and many variants, which are constantly being generated and selected against in a particular environment, as described earlier for RNA viruses and retroviruses.”  Comparing the adaptability of prions, a misfolded protein, to that of viruses, which bear genetic material and can respond to cellular changes with more plasticity, is fascinating.  The field strongly feels the changes in properties are most likely due to change in PrP^SC conformation.  

                Unfortunately, it also means that prions can develop drug resistance.  For this reason, many feel the best way to stop aggregate formation is to stop PrP synthesis or accelerate its turnover.  This idea has merit since PrP depletion in mice does not lead to devastating side effects.  However, the authors do end on the downer by saying that “no effective therapy is on the horizon.”

E. coli Infection

Reference

Zhang et al. “A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance.” Nature Chemical Biology (2011) 7, pgs 671 – 677

                For the Gram-negative bacterium Escherichia coli to successfully infect a victim following ingestion, it must survive a trip through the stomach and reach the small intestine.  Mammalian stomachs create a low pH environment to help break down incoming proteins from both food eaten and any accidently ingested pathogens.  Unfortunately, several enteric bacteria, including some strains of E. coli, are able to survive the acidic stomach to arrive at the neutral small intestine intact and successfully infect a victim.

                The outer membranes of Gram-negative bacteria are quite porous and will allow passage of molecules smaller than 600 Da.  Obviously protons can easily cross that membrane to reach the periplasmic proteins within.  How do the bacteria protect these proteins from either denaturation at low pH (stomach) or incorrect renaturation upon reaching neutral pH (small intestine)?

                It was previously known that the bacterial protein HdeA binds periplasmic bacterial proteins at low pH to protect them.  Once reaching the neutral small intestine, HdeA releases its substrates in a nonactive form that must then be properly folded again for full function.  What additional chaperones were involved in this process as well as substrates for HdeA were unknown.

                Recent work by Chen and colleagues, published last month in the journal Nature Chemical Biology, focused on identifying substrates for HdeA by using an unnatural amino acid (named DiZPK by the authors) whose side chain can photocrosslink with proximal protein.  They were able to place this version of HdeA inside living E. coli cells, subject them to low pH and thus identify substrates for HdeA.

                Interestingly, the two substrate proteins identified here are DegP and SurA, both of which are essential chaperone protein themselves.  The authors theorize that HdeA exists to protect these two important chaperones at low pH and helps refold them upon neutralization, which means they are then subsequently free to help other proteins refold (Figure 4.1, directly from their paper).  While the cytosol has mechanisms in place for chaperone protein folding mediated by ATP, the periplasmic space is low in ATP so the bacteria have developed another way to circumvent the situation.

                The importance of HdeA could lead to new therapies to treat E. coli infections.  

VEEV

Reference: Zhang et al. “4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus” EMBO (2011) 30(18), pgs 3854 – 3863.

VEEV = Venezuelan equine encephalitis virus

Fast Facts

-                Capable of infecting both humans and all species of equine (horses, zebras, donkeys)

-                Mosquito-borne pathogen

No human vaccine or antivirual drugs are available to treat VEEV.  Instead, an attenuated virus exists known as TC-83, which is given to laboratory workers and military personnel as a vaccine.  Because of its inability to be treated, high infection rate, and ease of production, VEEV has the potential to be used in bioterrorism.  In fact, the United States and a few other countries have developed VEEV as a biological weapon.  

In a recent issue of The EMBO Journal, Zhang et al. published the 4.4 Å electron cryo-microscopy structure of TC-83.  Partial X-ray crystallography structures were known of the viral coat proteins E1 and E2, but this data allowed researchers to determine reasonable models for both entire proteins.  

Figures 3.1 and 3.2 are taken directly from the paper and show the reported structure for one viral particle in 3D and a cross section of the virus.

Researchers say their data partially explains why TC-83 is attenuated compared with other VEEVs and offers insights on host recognition and initial nucleocapsid core formation.  For a virus we need to understand better, this structure and their work is definitely a step forward. 

The reference is above if you’d like to read more!

Double Duty Inhiitor?

REFERENCE: Hansen et al. Structure (2011) 19, pgs 919 – 929.

Malaria, a disease that causes 1 million deaths per year, is caused by a Plasmodium parasite.  Cysteine proteases (CPs) expressed by the parasite are implicated in key process of both parasitic life stages: liver and blood.  Interestingly, host cell CPs are also integral to infection.  Given the destructive nature of proteases, CPs of both host cells and parasites must be regulated site-specifically and temporarily.  In the July issue of Structure, Hilgenfeld and colleagues discuss the structure of the Plasmodium cysteine protease falcipain-2 (FP-2) in complex with the C terminus of their identified CP inhibitor from Plasmodium berghei (PblCP-C).  PblCP-C has an Ig-like ß sandwich fold and its closest structural relative is identified as chagasin, an I42 inhibitor family member.  Loops L0, L2, L4, and L6 protrude from PblCP-C (shown) into the active site of FP-2, thus occluding substrate binding.  The authors compare the PblCP-C:FP-2 structure to other solved inhibitor complexes and conclude that the major interactions responsible for inhibition are conserved between the structurally unrelated inhibitors, but the PblCP-C L0 interactions with FP-2 are unique to this complex.  Intriguingly, the structure of L0 also explains why PblCP-C is a potent inhibitor of the papain-like protease cathepsin L but not cathepsin B.  Because PblCP is necessary for host cell invasion, it is postulated that this CP inhibitor could block potentially deleterious protease activity at crucial moments, such as host-cell invasion, or inhibit host cell CPs (such as the cathepsin-like caspases).  It also provides a framework for developing small molecule inhibitors of the critically important FP-2.