T=3 plant viruses

Structural versatility gives quasi-equivalent packing in T = 3 plant viruses... 

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. 

One of the most striking results that has emerged from the high-resolution crystallographic studies of these icosahedral viruses is that their coat proteins have the same basic core structure, that of a jelly roll barrel, which was discussed in Chapter 5. This is true of plant, insect, and mammalian viruses. In the case of the picornaviruses, VPl, VP2, and VP3 all have the same jelly roll structure as the subunits of satellite tobacco necrosis virus, tomato bushy stunt virus, and the other T = 3 plant viruses. Not every spherical virus has subunit structures of the jelly roll type. As we will see, the subunits of the RNA bacteriophage, MS2, and those of alphavirus cores have quite different structures, although they do form regular icosahedral shells. 

Tomato bushy stunt virus is a T = 3 plant virus with 180 chemically identical subunits. Each polypeptide chain is divided into several domains. The subunits preserve quasi-equivalent packing in most contact regions by conformational differences of the protein chains, especially a large change in... 

The arrangement of subunits in the shell of picornavimses is similar to that of T = 3 plant viruses 334... 

Swelling that is dependent on divalent metal ions and pH is a common phenomenon in T=3 plant viruses, but it has not been observed in the picorna-like plant viruses or T=3 RNA animal viruses. The T=3 nodaviruses... 

Bromovirus—180 subunits in a 290-A-diameter T=3 icosahedral capsid Cozvpea chlorotic mottle virus Plants... 

Of the insect virus families, the capsid proteins of members of the Nodaviridae family are similar to those of plant viruses such an SBMV and TBSV, which have the same T=3 arrangement of subunits (Hosur et al, 1987). The capsid protein of the T=4 virus NudaureEa capensis ut (NwV) (Fig. Id see Color Insert), belonging to the Tetraviridae family, also has a jelly-roll topology, but the EF loop contains a complete domain of the immunoglobulin type c topology that is located on the outside surface of the capsid (Munshi et al., 1996). The N and C termini form a separate helical domain on the inside of the protein shell. 

Large-scale fluctuations of an animal virus particle are important for its ability to infect cells, but another type of particle dynamic behavior has been known for decades and was first identified in a plant virus. Brome mosaic virus (BMV), a single-stranded RNA (ssRNA) virus with T=3 icosahedral... 

The two highest concentrations of tannic acid (0.051 and 0.034%) resulted in a linear increase of virus titer up to 21 days after inoculation, even though the reduction of starch lesion formation was 91 and 64%, respectively. Thus, the virus must have replicated beyond the limitation of starch lesions. Further experiments indicated that a systemic spread of the virus into the primary leaves in cucumber plants could be obtained by daily brushing the noninoculated primary leaves (only the cotyledons were inoculated) with tannic acid following a vacuum infiltration of whole plants with 0.051% tannic acid 24 hours after virus inoculation. Primary leaves were shielded by tinfoil during the inoculation of the cotyledons to prevent accidental infection. Aerosol O.T. (0.1%) was incorporated in tannic... 

FIGURE 4.3 Schematic representation comparing the full and deconstructed virus vector strategies, (a) TMV expression vector, (b) Provector system for rapid assembly of viral amplicons in planta. P promoter, TMV Pol TMV polymerase, MP movement protein, GOI gene of interest, CP capsid protein, T terminator, RS recombination site. (Adapted from Gleba et al. (2004). Curr. Opin. Plant Biol., 7, 182-188.)... 

Cultures of PHA blasts are initiated by purifying peripheral blood mononuclear cells, usually by Hypaque-Ficoll density gradient centrifugation. These cells are then incubated with 10 pg/mL of the plant lectin phytohemagglutinin (PHA), which activates T cells. After 48 h, the medium is removed and fresh medium, supplemented with IL-2, is added. The cells are replenished with fresh medium and IL-2 every 3 d. These cells may be infected with either cell-free virus or with HIV-infected cells. Peak infection usually occurs anywhere from 7-28 d postinfection, depending on the size of the inoculum and the characteristics of the virus. In general, at the peak of infection, <5% of the cells in the culture are actually infected. However if extreme care is taken and the T-cell blasts are separated from nonactivated cells, it is possible to obtain infection in the majority of the cells in the culture (58). 

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