HSV-1 Electron Micrograph

HSV-1 GENOME MAP

                                                                                                                                                                 [47]
Gene Activation

    The core of HSV enters the nucleus, via a nuclear pore, where the genome is circularized. Transcription of the large, complex  genome is sequentially regulated in a cascade fashion. ~50 mRNAs are produced by host cell RNA polymerase II.
 
 

Genetic Variation
 

Phenotype

                                              Family:     Herpesviridea
                                              Subfamily:    Alphaherpesvirinae
                                              Genus:          Simplexvirus
                                              Symmetry:    Icosahedral (capsomers = 162) [6][43]

        Of all the Herpesviridea, the major herpes viruses infecting humans are herpes simplex 1 and 2. [7] All the Herpes Simplex Viruses (HSV) are thought to have evolved roughly in parallel with the vertebrate immune system, which  helps explain how it is well adapted for survival in its host. [78][63]  Relatively speaking both HSV-1 and HSV-2 have similar morphological structures, but biologically they are distinctly different. [7]   Both have a centralized dsDNA core surrounded by a capsid, the capsid is surrounded by a thick layer of additional proteins (Tegument) and an outer envelope with spike like glycoproteins.
 

 The Nucleocapsid shell is composed of these apparently hexameric units, composed of a principle structural protein VP5 (MW~155kd). The shell is approximately 175 angstroms thick with a radius 425-600Å (angstroms) {100-110 nm}. [11] A protein named VP23 (36 kd) is thought to connect the VP5 hexamers.[11]

Electron cryomicroscopy and computer image reconstruction of HSV-1

        In order to understand the structural mechanism of virus assembly and the molecular recognition and interactions between viral proteins and cellular receptors/ antibodies, Wah Chiu, Ph.D. has begun to analyses HSV-1 by electron cryomicroscopy and computer image reconstruction.  This was the preferred technique because the HSV-1 capsids are greater than 1250 Å in diameter. Up-to now they have been able to determine the structures of wild-type B capsids along with several recombinant capsids, to a resolution of ~13 Å. These resolutions  resolve not only the individual subunits in the hexons, pentons, and connecting proteins, but also the domain features for each of the four major structural subunits. By using different imaging techniques to compare the wild type capsids with the recombinant capsids lacking one capsid protein, the location of the missing proteins can be defined in the capsids and their biological roles can be deduced. Presently, the genes encoding the six known capsid proteins of HSV-1 have been identified and cloned into expression vectors. [41]

(a) A 26 Å 3D map of the T=16 icosahedral A-capsid of HSV-1 which is 1250Å in diameter viewed along 3-fold axis. One of the 20 icosahedral triangular faces is marked by solid lines. The adjacent pentons are separated by 675 Å. The reconstruction was done with 140 particles. The asymmetric unit consists of one penton subunit, one P-hexon, one C-hexon, half an E-hexon, and six types of connecting triplexes | Ta, Tb, Tc, Td, Te, and 1/3 Tf |. Both of these viruses share certain common assembly mechanism which requires the formation of icosahedral  procapsid shell of proteins in the presence of scaffolding proteins prior to the packaging of DNA. [41]

Illustrates the difference between penton and hexon subunits with an associated triplex. Isolated penton subunit with triplex Ta |left|, isolated P hexon subunit with triplex Tb |middle| in an equivalent orientation and their superimposed density maps |right|. The labels U, M, and L refer to the upper, middle, and lower domains of the penton subunits. The dashed lines in both the penton and hexon subunits denote the interpreted boundary between VP5 |150kDa| and the neighboring triplex subunit. In the superimposed map |right panel|, the hexon subunit and the associated triplex are depicted with contour lines instead of surface rendering. Both of the penton and hexon subunits are made up of VP5 and the horn-shaped domain in the hexon tip is interpreted as VP26 |12kDa|. [41]

(c) Display of Ta triplex. These are two legs |thick arrows| and an upper domain |dotted arrow| connected to a tail |::|. Both legs and the tail are connected to a floor of density with a central hole of 30 Å in diameter. The thin arrows point to the two arms connected to the adjacent VP5. The two legs are interpreted as VP23 |36kDa|. The tail and upper domain are interpreted as VP19c |57kDa|. These results are described in more detail in a recent paper of Zhou et. Al.  [41]

HSV-1 -capsid from 400kV Spot-scan Electron Cryomicroscopy     [54]

                Published on the Internet. Reproduction granted for educational purposes. [54]

 3-D computer reconstruction from cryo-electron micrographs
 

gD Glycoprotein

        Because of the overall diversity of gD's functions, it is important to try to understand gD's structure in greater detail. gD is the most essential glycoprotein for HSV's entry into mammalian cells. Beside penetration, gD has been implicated in cell fusion, cell to cell spread, super infection and neuroinvasiveness. [12] In cell to cell spread it has been demonstrated that gD, gE, and gI as essential but gE and gI are not needed for entry of free virus. [14]

        At the amino acid level, gD from HSV-1 (gD-1) is 85% identical to its homologue in HSV-2, gD-2. The two proteins are functionally interchangeable and give rise to type common and type specific monoclonal antibodies. [12] Antigenic, biochemical, and mutational analyses have led to a current model of gD structure.  In the extracellular portion of gD there are three di-sulfide bonds which are necessary for the stability of the structure of gD as well as the function of the virus. [12] (see Image)  Both gD-1 and gD-2 have identical di-sulfide bonding patterns and both gD-1 and gD-2 exhibit high structural and functional homology. [12] In order to define functional domains of gD,  A.V. Nicola, et.al  used linker insertion mutations. The properties of the insertion variants indicated that 4 separate regions of gD are important for function. [12] These were defined as region I (residues 27-43), region II (residues 126-161), region III (residues 225-246), and region IV (residues 277-310). (see Image)  These studies showed that despite changes in gD structure and stability, all mutant proteins bound to cells, suggesting that binding is not the only function of gD.  However, the region I mutants failed to inhibit HSV plaque formation and cell-cell spread. In contrast it was discovered that the region II, III, IV mutants do inhibited these processes, indicating their possible functional roles. Thus, the ability of gD to bind to cells and inhibit infection does not correlate with its ability to initiate infection. [12] Not only does this data suggest that gD has more than one functional domain but these studies also confirm that gD-1 and gD-2 are structurally similar and differences can be demonstrated.  By using a quantitative ELISA differences in the reactivity of gD-1 and gD-2 with type common Monoclonal Abs, suggesting that shared epitopes are presented somewhat differently by the two proteins which has significant implications in vaccine development. (Vaccines)Also,  it was found that both proteins are primarily beta-sheet in secondary structure. However gD-1 had more alpha-helical content than gD2 and gD-2 had more beta-sheet content than gD-1. [12] Related studies have shown that glycoproteins gB, gD, gH, gL are essential for penetration to host cell and for cell to cell spread. The three N-linked oligosaccrides are dispensable for gD function in vitro and in vivo but critical for the maintenance of antigenic structure. [12]
 

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