Vesicular Stomatitis Virus (Rhabdoviridae family)
Created by Husayn Tavangar
Vesicular Stomatitis Virus (VSV) (PDB ID: 2J6J) is a virus in the Rhabdoviridae family. It carries its genetic information in the form of a single stranded minus sense RNA. It is enveloped, bullet shaped and 175 nm in length and 65 nm in width1. Insects (sand flies, black flies) are vectors for the virus, spreading it to animals that they bite, so VSV can be classified as an arbovirus. It can affect horses, donkeys, mules, cattle and swine. This viral infection is of economic consequence to farmers because when one animal gets infected, the virus usually spreads to the whole herd. VSV causes vesicular stomatitis, which is characterized by blisters on nostrils, lips, tongue and gums of livestock. Blisters can also be found on teats and hooves. These blisters are very painful for the animal, causing refusal to eat and weight loss. Blisters on teats reduce milk production in dairy cows and blisters on hooves can cause lameness in any infected animal. Even though VSV infection is rarely fatal, large drops in productivity ensue. There is no cure or vaccine available so the way to deal with VSV infection is prevention and quarantine. Humans can be infected by VSV, resulting in flu-like symptoms.
The VSV G protein allows viral entry into cells. It binds to host cell (unknown receptor) and is then endocytosed. G then causes fusion of the viral envelope with the endosomal membrane. The VSV fusion pathway has discrete steps. First, the virion associates with the target membrane via its flat base. Second, the local membrane deforms, forming stalks and fusion pores. Conformation changes in glycoprotein at lower pH lead to enlargement of these pores and membrane fusion. Glycoprotein, located outside the contact zone between virions and liposome, reorganizes into regular arrays. The formation of these arrays might induce membrane constraints, achieving the fusion reaction. There are 2 forms of G: a pre and a post fusion conformation. These two forms are present in equilibrium. The conformation required for membrane fusion is pH dependent because when pH is not low enough (below pH=5.5), fusion does not occur2. Also, there is evidence that VSV promotes its own uptake by inducing clathrin coat formation3. The clathrin coat forms near the virus particle, possibly indicating that G or its receptor might somehow recruit the adaptor complex AP-2. Another interesting finding is that the fusion of VSV and the subsequent release of the viral nucleocapsid into the cytosol occur sequentially, at successive steps of the endocytic pathway4.
I would like to compare the VSV G protein with Glycoprotein B from Herpes simplex virus type 1, low-pH (PDB ID: 3NWF)5. HSV-1, which produces most cold sores in humans, is ubiquitous and contagious. Because of its high prevalence, 57%6, and no cure, HSV-1 is an important virus to understand. First difference between the 2 proteins is that the VSV G is a 1 chain polypeptide (which trimerizes)while the HSV G protein is a homotetramer. Both proteins have a similar overall structure: Both consist of a main globular body with three arm-like extensions, which all point in the same direction (like a three legged table). In both cases, the arms are made of mostly β-strands. However, the tips of the HSV-1 arms have more α-helices than the arm tips in VSV G. Also, the HSV-1 G has an additional larger arm sticking out from the globular central mass. This arm points in the opposite direction to the other 3 arms.
The amino acid sequences of HSV G and VSV G were compared using the blastp “align two sequences” option. There were 3 regions of matching. The largest region of matching was between residues 449-541 of HSV-G and 307-391 of VSV G. This region consisted of many conserved P and N residues. We can conclude that this region does not represent an α-helix in VSV G. A side by side comparison of these two conserved regions in the two polypeptides gives us a better picture of what region of the protein is conserved. The second region of matching was between residues 80-104 of HSV G and 15-47 of VSV G. This region had 2 conserved N and P residues. The third region of matching was from 7-22 of HSV G and 306-321 of VSV G. There were 2 conserved P residues and 1 conserved N residue. These conserved amino acids must be important in providing the virus with an evolutionary advantage. The conserved amino acids are probably involved in critical functional roles, like interacting with receptor on host cell membrane. The many conserved P and N residues are of interest.
The VSV G protein crystallographic structure was elucidated in 20077. The method used was X-ray diffraction. The structure of G was determined to 3.0 angstrom resolution. More specifically, Roche et. al, determined the structure of the prefusion form of the VSV G protein ectodomain (PDB ID 2J6J). The G fragment consists of 422 residues (residues 17-438 is ectodomain), weighs 48324.19 Da and is 88 angstroms in length. The secondary strucuture of G is a mixture of alpha helices, beta sheets, 3/10 alpha helices and random coils. G is a trimeric transmembrane protein. The overall architecture of G resembles a tripod. In this tripod organization, the fusion domains are spread apart, keeping the fusion loops separate. G is composed of the following domains: Trimerization domain (residues 18 to 35, 259 to 309 and 383 to 405), Lateral domain (residues 1 to 17 and 310 to 382), PH domain (residues 36 to 46 and 181 to 258), Fusion domain (residues 53 to 172), C-terminal part (residues 406 to 413) and the Rigid block or RbI-II (residues 1 to 25 and 273 to 382). The fusion domain is of particular interest because this is what inserts into the host cell’s endosomal membrane, allowing viral entry into the cell. This structure can be used to aid drug delivery into cells7. It is composed of multiple β-sheets and 1 α-helix. For the portion of the fusion domain that penetrates a membrane, we expect to find hydrophobic residues to ease the passage through a lipid bilayer. Indeed, we see that the hydrophobic residues Y116, A117, W72, and Y73 are exposed at the end of the fusion loops.
Protonation of His residues H-407, H-60 and H-162 at low pH (environment of endosome) can contribute to destabilization of the interaction between the C-terminal segment and fusion domain in the prefusion G. The conformation change due to His protonation can facilitate the fusion domain approach toward the target protein. In the postfusion conformation, acidic residues D-268, D-274, D-395, E-276 and D-393 were sequestered in some way. The residues were sequestered either by burial at trimer interface (D268) or by bunching together (D274 with D395 and E276 with D393). In the prefusion conformation, these same acidic residues are solvent exposed. These pH dependent residues bring in the idea of pH-sensitive molecular switches, a common method for protein function modulation. His residues in the prefusion conformation and acidic residues in the postfusion conformation (mentioned above) are the pH-dependent molecular switches of G.
In 2006, before the elucidation of the prefusion G protein, the same research team determined the crystal structure of the VSV G protein under its acidic conformation8 (PDB ID 2CMZ). This form corresponds to the G’s post-fusion conformation. They determined both of these structures to try and find out what conformation changes occur in the protein during membrane fusion. These conformational changes are important because they enable viral entry into the host cell. By observing what domains change or move during fusion, we can conclude that these changing domains are functionally important. During the transition from the pre to post fusion conformations both the fusion loop and the TM domain move 160 angstroms from one end of the molecule to the other. In other words, both the fusion domain and the TM segment are flipped relative to RbI-II. The fusion domain is projected toward a target membrane by the combination of 2 movements. First, a 94° rotation around the hinge between the fusion and PH domains occurs. Second, the PH domain is reoriented at the top of the trimerization domain. Residues 47 to 52 and 173 to 180 are reorganized. The helix in the first segment is unfolded while a helix is formed in the second segment. Also, major conformational changes in the trimerization domain (where the three protomers associate) occur. Refolding in the trimerization domain is critical because this drives the repositioning of the PH domain and the flipping of the C-terminal part. Some conformational changes are: central helix residues 276 to 294 is lengthened by the recruitment of a segment (made up of residues 263 to 275) to form a longer helix and residues 384 to 400 refold into a helix.
When thinking about a treatment for VSV infection, viral antigenic sites are of importance as potential ways to vaccinate against the virus. These antigenic sites have been identified by comparing the VSV G sequence to that of the rabies virus. Some of the corresponding antigenic residues are 37-38 and 341-347. The surface of α-helix E is also an antigenic site. During the prefusion to postfusion conformational change, only the 341-347 antigenic site is preserved.