Monday, 4 August 2014

towards a high-resolution understanding of hepatitis C virus

HCV virions
Viruses are sub-microscopic parasites of cells. What this means is that despite our immense interest in them, they are very difficult to observe and study using your eyes or even traditional light microscopy, which is the mainstay of biology over the last few centuries. This is annoying because we are interested in understanding what viruses look like because of what they tells us about their biology as virus particle structure affects many aspects of its lifecycle, such as cell entry, replication and transmission. 

These questions are no more interesting than for HCV, an important human pathogen that has chronically infects between 130 and 150 million people worldwide and leads to between 350,000 and 500,00 deaths a year. Infection often lead on to chronic hepatitis, cirrhosis, fibrosis, hepatocellular carcinoma and eventually liver failure and death. Although there are now effective drugs targeting HCV, HCV and associated diseases are likely to continue to be a public health issue in the future because of their prohibitive cost. We do not yet have an effective vaccine to protect against the virus either. 

One avenue to aid development of antivirals and vaccines is to understand HCV particle (virion) structure and the pathways that promote its assembly and its entry into cells. The catch is that HCV has been extremely challenging to manipulate in the lab under experimental settings. However, a paper published in the journal PNAS, (free here from the Charles Rice lab in New York (first author: Maria Teresa Catanese) has shown the characterisation of HCV virion structure using a powerful microscopic technique: cryoelectron tomography (cryo-EM), which has improved our understanding of HCV biology. The continuing use of this imaging technology combined with models of HCV entry and assembly may aid in the development of novel HCV drugs and vaccines. 

HCV virion structure

Like many other viruses, HCV virions were initially showed to be composed primarily of a nucleic acid genome (RNA is this case) encased within a protein shell and a lipid 'envelope'. This envelope is covered in glycoproteins, which facilitate binding to host cells and virus entry. This entire structure allows for the efficient protection of the virus genome as well as its assembly, release and subsequent entry.

An intriguing feature of HCV is its variable buoyant density of infectious particles taken from patient sera and tissue culture supernatant, which is strikingly lower than other RNA viruses. Infectious HCV buoyant density looked more similar to that of human sera lipoproteins that to other viruses. For example, measles virus has a buoyant density of 1.23 in caesium chloride, which is also similar to influenza virus in sucrose. Even very closely related viruses to HCV, such as the pestiviruses and the flaviviruses had buoyant densities much higher. Even, liver-tropic viruses such hepatitis B virus did not have densities this low. This suggested that something else, specific to HCV. Early on in HCV research it was demonstrated that infectious particles associated with human serum lipoproteins. And it is this that might explain some key features of HCV biology and pathogenesis.

Lipoproteins are lipid and protein complexes, mainly synthesized in the liver, which can aid the movement of fats around the cell and body by the proteins emulsifying the fats and hence making them soluble in water. Their structure also aids in the regulated delivery of lipids to cells like liver, muscle and fat cells. The general structure of these particles is that of roughly spherical with the hydrophilic regions aimed outwards while the hydrophobic, fatty parts are buried deep inside. One of the most well-known classes of lipoproteins are the high (good cholestoerol) and low (bad cholesterol)-density lipoproteins (H-/L-DLs). These assemblages are complex and often are composed of many different kinds of proteins and lipids. Of interest to HCV research, lipoproteins such as LDL, intermediate (I) DL and very low (VL) DL have buoyant densities within the same range as the observed infectious HCV particles, suggesting that HCV could be interacting directly with them.
a cartoon lipoprotein complex (wikipedia). Cholesterol and triglycerides are found internally to phospholipids and proteins. 


What we knew before was that the HCV virion was complicated but very interesting from a scientific point of view and that it had proved to be elusive when trying to study it using the best microscopic techniques we had, for example, cryo-electron micoscopy and tomography. Cryo-EM is a way to study particles, such as viruses, in a their almost-native state following freezing. Frozen suspensions of particles are bombarded with electrons to form an image. Multiple images are interrogated to assemble single particle 3-D reconstructions.
purification of HCV virions

The paper of Catanese et al., took advantage of being able to grow HCV in the lab in liver cancer cell lines and modified primary human hepatocytes and were able to infect these cells in the lab and remove the replicated HCV released into the cell supernatant. Once removed, HCV virions were purified using grids, coated with antibodies that specifically recognised and bound to HCV (note that the best way to get purified HCV was to use a genetically engineered HCV that expressed a high-affinity protein tag on its surface). These purified virions were then subjected to cryo-EM to observe exactly what they look liked, in a way that we had never before been able to achieve. The show some very nice pictures of purified HCV and are able to quantify a number of basic biological features of the virus.

HCV single particles by cryo-EM
Using cryo-EM they were able to show that while HCV particles were generally spherical, their size was quite variable (2.5 fold (40nm to 100nm), which is unlike other related viruses. Catanese et al., were able to visualise HCV interaction with lipoprotein complexes. Like other studies before them they confirmed the presence of certain apolipoproteins and interestingly that these lipoproteins may be preventing binding of antibodies to HCV glycoproteins. This has implications for HCV vaccine design.

Only the start

I believe that this work (representing a technological advancement) is only the start of improving our understanding of HCV structural biology and how it relates to infection and pathogenesis. Improving upon these data and combining them with crystal structure of purified HCV glycoproteins, for example, should provide further knowledge of HCV structural biology. A test of this technology would be to image HCV from clinical material over the course of an infection, but I can see how difficult that may be. However, combining high-resolution imaging (EM and confocal microscopy combined) investigations with hypothesis-driven molecular biology could tease apart the mechanisms of HCV entry, assembly and spread in clinically-relevant model systems (humanised mice). Understanding these mechanisms may facilitate targeted therapeutics and vaccine development.

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