NIAID-DVI: The Dynamic Dengue Particle -- Richard Kuhn

New Lessons in dengue virus structure and composition and their influence on vaccine strategies
Richard Kuhn
Purdue University

I had the honor and pleasure of meeting Dr. Kuhn where I work and he is fantastically animated and I very much enjoyed listening to what he had to say.

The structure of dengue has been known for more than 10 years via cryo-electron microscopy; however now with more sophisticated tools we can see the virus in ways unimaginable in the past. His group employed a variety of structure and biochemical tools to probe the structure of the dengue virion as well as conformation, composition and dynamics..

Lets look at some particles...

mmm...pretty. Clustering of dengue particles. Source: MicrobiologyBytes

• Cryo-electron microscopy (Cryo-EM) shows that virions come in a range of immature, mature and 'somewhere in between' particles.
• Mature particles lack prM
• Temperature influences ratios of mature to immature particles
• Cell type as we  learned in the previous blog also influences the ratio of mature to immature particles.
• Mature particles can be thought of as 'smooth' while immature particles can be thought of as spiky.

Cryo-EM: Immature (source)

Cryo-EM: Mature (source)

Some other cool pictures:

Immature (spiky), Source

Mature (smooth), Source

So what happens to make the particle go from an immature 'spiky' state to a mature 'smooth' state?

 A model of flavivirus maturation pathways. The pr/prM proteins are shown in red/blue and the envelope proteins are shown in grey. The red and blue colours of prM indicate mismatched icosahedral symmetries. ER; endoplasmic reticulum; pr, precursor; prM, precursor membrane; TGN, trans-Golgi network. From Plevka et al., 2011

• Immature particles form by budding into the endoplasmic reticulum. 
• The envelopes of these particles have a single icosahedral symmetry. 
• A conformational change of the virion occurs as the particles are transported to the acidic pH in the Golgi and TGN. 
• The prM protein can be cleaved by the host protease furin after the conformational change, but not all prM molecules are cleaved sometimes. Depending on the fraction of prM cleaved, the particles might adopt mature (smooth), mosaic (spiky/smooth) or immature-like (spiky) conformations when released from cells. 
• The cleaved precursor proteins are released from particles at neutral pH into the extracellular space. 

Note: bullets taken from Plevka et al., 2011 text.

So it is the incomplete or lack of cleavage of prM that gives us the different phenotypes of dengue viral particles.

Unfortunately Kuhn's work is pretty much all under a paywall...boo hoo, that kind of hoses me; but in my desire to provide more awesome pictures, I've combed the net so as to not disappoint...I've linked the articles in the references at the end of this blog, but unless you have a subscription your pretty much sh*t out of luck, like myself.

One last picture I was able to find from one of Kuhn's group's work on temperature differences and how they affect particle maturation:

Zhang et al., 2013

I found an article in Nature Structure and Molecular Biology available on ReadCube, huzzah...that had a run down of the study on dengue maturation and temperature, referencing Zhang's work.

From 'Dengue Likes It HOT' (Source):

• Several epitopes recognized by neutralizing monoclonal antibodies are not exposed in the smooth, mature form of the particle at 20oC.
• When mature virus was incubated at 37oC (human temp) it took on the bumpy appearance exposing the epitopes but not becoming the 'spiky' immature form...they were 'in the middle'
• The immature form stayed spiky at 20oC and 37oC
• Transition from mature smooth to the mature bumpy particles happens between 31-35oC and once this change takes place it is irreversible.
• Cryo-EM of the mature 'bumpy' 'warmer' particle showed a different arrangement of the dimers.
• Preliminary data suggests these 'mature bumpy' particles are more infective as shown in cell culture but this hypothesis needs to be tested further.

So the Modis research group has also been working on the E gene structure and particle formation for a while as well. From here we will delve into the 'hinge' work that's been done and was also mentioned briefly in my previous blog.

• There is a hinge between domains I and II of the E gene can range in 'degree' from 6 to 27 depending on whether its the mature virus or if we are looking at the crystal structure. (Zhang et al 2005, Mukhopadhyay et al., 2005)
• The E protein undergoes conformational changes during assembly of the virus particle and fusion causing a 30 degree shift in the hinge region between domains I and II. (from ALFP Medical Article on Dengue virus; quite good!)
• Modis' group describes the hinge as a hydrophobic pocket the promotes domain 'flexing'. (Modis et al., 2003)
• Both groups Modis' and Rossmann's group which solved the structure of dengue found similar structures except for the hinge 'motion' between domain I and II which was different by 10 degrees (from ALFP Medical Article on Dengue virus).

Viral Structure and Hinge:

  Semitransparent surface representation of the dengue virus E protein. The two subunits in the dimer are shown in different shades of blue. The backbone of the molecule is superposed in a yellow ribbon representation. The two glycosylation sites are indicated. The sugars linked to Asn-67 and -153 are shown as yellow and red sticks, respectively. The β-octyl glucoside molecule (indicated by bOG) is displayed as spheres colored according to atom type (red, oxygen; gray, carbon). It lies in a pocket at the hinge region between domains I and II. Figure prepared with the program ribbons. (From: Rey 2003)

SO...to Sum Up:

1. Dengue has several forms: mature, immature, mosaic (mature/immature mix) and mature yet bumpy
2. The hinge regions plays a key role in conformational changes necessary for membrane fusion and transition from the immature to the mature viral form.

You can imagine what amino acid changes within the hinge region might do then, impeding or changing the way the hinge bends thereby affect viral fusion and maturation.

We've only touched the tip of the iceberg and there is more to come I am sure from many groups on dengue viral structure and dynamics.

Kuhns Group or Involvement Cites:

• Kuhn et al., 2002. Structure of dengue virus: implications for flavivirus organization, maturation and fusion. Cell 108: 717. (I found the pdf, huzzah!)
• Li et al., 2008. The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science 319: 1830. (paywall)
• Yu et al., 2008. Structure of the immature dengue virus at low pH primers proteolytic maturation. Science. 319: 1834. (paywall)
• Zhang et al., 2013. Dengue structure differ at the temperature of its human and mosquito hosts. PNAS 110: 6795. (damn I wish this wasn't paywalled)

Modis' Group Cites:

• Modis et al., 2005. Variable surface epitopes in the crystal structure of dengue virus type 3 envelope glycoprotein. JVI 79: 1223. (free! huzzah!)
• Modis et al., 2003. A ligand-binding pocket in the dengue virus envelope glycoprotein. PNAS 100: 6896. (free)
• Modis et al., 2004. Structure of dengue virus envelope protein after membrane fusion. Nature. 427: 313. (paywall)

Some other cool articles on immature/mature dengue and implications thereof: 

• Rodenhuis-Zybert IA, van der Schaar HM, da Silva Voorham JM, van der Ende-Metselaar H, Lei H-Y, et al. (2010) Immature Dengue Virus: A Veiled Pathogen? PLoS Pathog 6(1): e1000718. (I love that PLoS is always free!)
• da Silva Voorham JM, Rodenhuis-Zybert IA, Ayala Nuñez NV, Colpitts TM, van der Ende-Metselaar H, et al. (2012) Antibodies against the Envelope Glycoprotein Promote Infectivity of Immature Dengue Virus Serotype 2. PLoS ONE 7(3): e29957