Of the virion

The shell of all picomaviruses is built up from 60 copies each of four polypeptide chains, called VPl to VP4. These are translated from the viral RNA into a single polypeptide, which is posttranslationally processed by stepwise proteolysis involving viraily encoded enzymes. First, the polypeptide chain is cleaved into three proteins VPO (which is the precursor for VP2 and VP4), VPl and VP3. These proteins begin the assembly process. The last step of the processing cascade occurs during completion of the virion assembly the precursor protein VPO is cleaved into VP2 and VP4 by a mechanism that is probably autocatalytic but may also involve the viral RNA. VPl, VP2, and VP3 have molecular masses of around 30,000 daltons, whereas VP4 is small, being 7000 daltons, and is completely buried inside the virion. 

The asymmetric unit contains one copy each of the subunits VPl, VP2, VP3, and VP4. VP4 is buried inside the shell and does not reach the surface. The arrangement of VPl, VP2, and VP3 on the surface of the capsid is shown in Figure 16.12a. These three different polypeptide chains build up the virus shell in a way that is analogous to that of the three different conformations A, C, and B of the same polypeptide chain in tomato bushy stunt virus. The viral coat assembles from 12 compact aggregates, or pen tamers, which contain five of each of the coat proteins. The contours of the outward-facing surfaces of the subunits give to each pentamer the shape of a molecular mountain the VPl subunits, which correspond to the A subunits in T = 3 plant viruses, cluster at the peak of the mountain VP2 and VP3 alternate around the foot and VP4 provides the foundation. The amino termini of the five VP3 subunits of the pentamer intertwine around the fivefold axis in the interior of the virion to form a p stmcture that stabilizes the pentamer and in addition interacts with VP4. 

Choi, H.-K., et al. Structure of Sindbis virus core protein reveals a chymotrypsin-like serine proteinase and the organization of the virion. Nature 354 37-43, 1991. 

The development of AAV-based gene-therapy vectors is presently focused on the analysis of the properties of the different AAV serotypes, especially the host range of the virion shells of the different serotypes. By packaging... 

Gibson W (1996) Structure and assembly of the virion. Intervirology 39 389 00 Goldman ME, Nunberg JH, O Brien JA, Quintero JC, Schleif WA, Freund KF, Gaul SL, Saari WS, Wai IS, Hoffman JM et al. (1991) Pyridinone derivatives specific human immunodeficiency virus type 1 reverse transcriptase inhibitors with antiviral activity. Proc Natl Acad Sci USA... 

Okada, T., Patterson, B.K., Ye, S.Q. and Gurney, M.E. (1993) Aurothiolates inhibit HIV-1 infectivity by gold(I) ligand exchange with a component of the virion surface. Virology, 192, 631-642. 

Enveloped viruses Many viruses have complex membranous structures surrounding the nucleocapsid. Enveloped viruses are common in the animal world (for example, influenza virus), but some enveloped bacterial viruses are also known. The virus envelope consists of a lipid bilayer with proteins, usually glycoproteins, embedded in it. Although the glycoproteins of the virus membrane are encoded by the virus, the lipids are derived from the membranes of the host cell. The symmetry of enveloped viruses is expressed not in terms of the virion as a whole but in terms of the nucleocapsid present inside the virus membrane. 

Attachment (adsorption) of the virion to a susceptible host cell ... 

Fig. 5.3 Destruction of virions of influenza under irradiation in the presence of suspension and Or (a) Intact virions in allantoic fluid and (b) virions after 5h of irradiation in presence of oxygen. Note the fragmentation of the outer membrane of the virions and the loss of a significant part of surface glycoproteins. Negative contrast, bar lOOnm...

Morphological studies show that SFV particles bound to BHK-21 cells are preferentially associated with the microvillar projections of the cell surface membranes (Helenius et al., 1980). Many of the virions which are not bound to microvilli (5% of all the cell surface viruses) are located in coated pits. The coated pits are invaginations of the plasma membrane, with a characteristic electron-dense coat composed of clathrin and other proteins on the cytoplasmic face (Pearse and Bretscher, 1981). Many of the coated pits are localized close to the base of microvilli. 

The reverse transcriptase enzyme (RT) is the primary enzyme responsible for the conversion of the viral single-strand RNA to the double-strand DNA. The reverse transcriptase enzyme is a component of the virion and is encoded by the pol gene. The RT is manufactured in the HIV-infected cells as a gag-pol fusion polyprotein. The RT is not the only enzyme necessary for the translation of RNA to DNA. The other enzymes for this conversion include RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H (Gilboa and Mitra, 1978 Prasad and Gogg, 1990). The reverse transcriptase enzyme has a high error rate (1 in 2000 bases), which produces higher incidents of mutation. Some of these mutations make the virus resistant to NNRTI treatment. 

Docosanol is not directly virucidal instead, it blocks the entry of the virion into the host cell by inhibiting the fusion of the viral envelope with the host plasma membrane. Because it does not affect viral replication or protein production, it may be less susceptible to the development of resistance than other antiviral drugs. 

HIV protease is an homodimer of C2 symmetry. This enzyme specifically cleaves the precursor polyprotein and affords the proteins required for maturation of the virion and for virus replication. Some pseudosymmetric inhibitors have been conceived on this basis. For example, pseudosymmetric difluoroketones exhibit subnanomolar inhibitions and are active toward infected lymphocyte MT-4 cell lines. Nevertheless, simpler a-peptidyl fluoroketones are also good inhibitors (Figure 7.41). The difluorostanones developed by Merrell-Dow were very efficient toward infected cells. ... 

Figure 9.1 The viral infectious unit, or virion, consists of a nucleic acid encased in a protein shell. The various structural components of the virion exhibit differing functional properties and thus afford a variety of targets for antiviral drug design.

There are between 50 to 100 neuraminidase spikes per virion [36] which is approximately 10% of the visible spikes projecting out of the surface of the virion [37]. These spikes can be removed from the vims by treatment with detergent [38]. Electron microscopic images of the neuraminidase spikes [39] reveal a mushroom-shaped molecule made up of a boxlike head of about 80 x... 

The VP1, VP2, and VP3 all contain a core eight-stranded antiparallel (3 barrel (Figure 2). These polypeptides have surfaces on both the exterior and interior of the virion particle, facing both solvent and viral RNA. The fourth polypeptide, VP4, is considerably smaller than the others and does not contain the eight-stranded barrel motif. The VP4 resides entirely on the interior surface of the virion, in close association with the viral RNA. The N-terminus of VP4 is known to be myristoylated in both rhino-andpolioviruses [18,19]. 

Schematic illustration of the icosahedral rhinovirus 14. (a) Shown is the icosahedron comprised of 60 copies each of VP1 (light gray), VP2 (black), and VP3 (gray). The shaded circles around each five-fold axis indicate the canyon positions. Also indicated is the approximate position of the VP1 hydrophobic pocket that lies underneath the surface of the virion, (b) An icosahedral pentamer is expanded with one viral protomer shown as a protein ribbon diagram, (c) This pentamer is seen in a cutaway view. Here VP1 is white, VP2 and VP4 black, and VP3 gray. A capsid-binding compound is depicted as black spheres inside the VP1 ribbon diagram. The cross hatched regions on the (c) schematic (right) indicate areas that disorder when HRV14 crystals are exposed to acid.

The formation of these types of particles can be induced by association of the virion with the cell receptor or acidification [32,34]. In poliovirus, attachment to cells has been shown to lead to a particle that has lost VP4 and has externalized the N-terminal region of VP1, which normally resides on the virion interior. 

VP4 contains the myristoyl moiety, which can signal VP4 to associate with a membrane. The VP4 would drag the N-terminal region of VP1 (to which it is closely associated) to the exterior of the virion. This would result in a particle with the N-terminus of VP1 exposed, as has been observed in poliovirus [48-50]. The sequence of this exposed region of VP1 suggests that it can form an amphipathic helix. 

The structure of the first compound to be solved in complex with HRV 14 was found to be bound in an extended conformation within the VP1 hydrophobic pocket [24]. The compound is almost entirely buried within the capsid of the virion. Since in the native HRV14 the pocket exists in a closed configuration, binding required large motions in VP1 to accommodate the drug. These... 

Three nearest neighbors, indexed as 0, 6, and 11, are indicated. The axial slab shown represents 1% of the total length of the virion. From Marvin.31a (B) A 2.0 nm section through the virus coat with the helices shown as curved cylinders. The view is down the axis from the N-terminal ends of the rods. The rods extend upward and outward. The rods with indices 0 to -4 start at the same level, forming a five-start helical array. The rods with more negative indices start at lower levels and are therefore further out when they are cut in this section. (C) The same view but with "wire models" of the atomic structure of the rods. From Marvin et al 2... 

Success in treating AIDS may depend upon better understanding of the complex life cycle of HIV-1,722,730,735 w -,jc -, js summarized in Fig. 28-27. The cycle begins with the binding of the virion envelope protein to the immunoglobulin-like surface protein CD4, which is found principally on the type T4 helper T cells (Chapter 31). Binding of CD4 to the HIV envelope proteins appears to activate the T cells to enter the cell cycle and to take up and integrate the virus. The virus infection destroys these CD4+ lymphocytes with a half-life of less than two days.735... 

Several methods of viral classification are in use. Classification based upon epidemiological criteria, such as enteric or respiratory viruses, is useful, but of more significance are schemes based upon the morphology of the virion (symmetry, envelope, etc.) and lype of nucleic acid (DNA, RNA, number of strands, polarity, etc.)... 

The protocol involves a classical SDS-PAGE (10% polyacrylamide) run, followed by transfer onto a Western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be taken during sample preparation, because phages are very stable and difficult to denature. The protocol is similar to typical SDS-PAGE sample preparation, except that / -mer cap toe thanol should be replaced by fresh dithiothreitol (DTT, 5 mM final concentration), and the samples should be boiled in a water bath for at least 15 min. Moreover, because the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded onto the gel, typically around 1012 phages per lane. 

Influenza virus sialidase poses a highly attractive target for antiviral drug design due to its prominent position at the surface of the virion and its profound role in viral pathogenesis. Random screening programs identified a number of... 

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