BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 30. VIRUSES

30.3. Protein Disks Join an RNA Loop during TMV Assembly

A priori, the simplest mechanism for TMV assembly would be the stepwise addition of individual protein subunits to the RNA. The difficulty with this approach, however, is that the nucleation rate would be extremely low. Before the complex could close upon itself to form one turn of the helix and thereby stabilize, approximately 17 coat subunits would have to attach to the flexible RNA molecule. This problem can be overcome by the simultaneous addition of a multi-subunit complex to the RNA. Indeed, the coat protein readily forms a double-layered disk consisting of 34 subunits. Each layer of the disk is a ring of 17 subunits; approximately this number of subunits (16 ¹/₃) corresponds to one turn of the TMV helix. Klug and his coworkers investigated the three-dimensional Structure OF THE disk (Fig. 30.5) and showed that it serves as the main intermediate in TMV assembly. A crucial property of the disk is that its subunits can slide relative to one another to form a two-turn helix, the so-called "lockwasher" (Fig. 30.6).

Class="center">Fig. 30.5. Electron density map of the TMV protein disk. A layer 6 Å thick is shown

Fig. 30.6. Schematic of The conversion of the TMV protein disk into the helical "lockwasher" form

The disk interacts much faster with TMV RNA than with foreign DNA. Consequently, TMV RNA evidently contains a base sequence specifically recognized by the disk that initiates assembly. This initiation region was isolated as follows. Several disks were added to TMV RNA to cover the initiation region, and the remaining RNA was then digested with a nuclease. The protected fragment contains approximately 65 NUCLEOTIDES that bind tightly and specifically to the disks. The base sequence in this initiation region strongly suggests that this RNA fragment forms a hairpin structure with a base-paired stem and a loop (Fig. 30.7). Most interestingly, the loop contains a G residue at every third position. This arrangement of bases in triplets corresponds to the Stoichiometry of the viral coat subunits—one subunit per three nucleotides. Therefore, the loop most likely binds to the first disk, initiating the assembly of the viral particle.

Fig. 30.7. The TMV RNA region responsible for the initiation of TMV particle assembly

Unexpectedly, the initiation loop was found to be located far from both ends of the RNA. The assembly start site is situated 5,300 nucleotides from the 5' end and approximately 1,000 nucleotides from the 3' end of the RNA. Another surprise was that both ends of the RNA emerge from the same side of the growing TMV particle (Fig. 30.8). The length of the 3' end remains more or less constant throughout the assembly process, whereas the 5' end becomes shorter as the viral particle elongates.

Fig. 30.8. Electron micrograph of partially reconstituted TMV particles. Two RNA tails are visible extending from each growing virion

The most probable model for particle formation is illustrated in Fig. 30.9. Assembly begins with the insertion of the initiation loop into the central channel of the double-layered protein disk. The loop binds to the first turn of the disk, and the adjacent base-paired stem unwinds. This interaction converts the disk into the helical lockwasher form, and the disk traps the RNA. Thus, the building of the viral helix begins. Next, another disk attaches to the newly formed RNA loop protruding from the central channel. As each subsequent disk is added, a new loop is formed by pulling the 5' end through the central channel of the growing viral particle. Finally, the 3' end is encapsulated in a manner not yet fully understood.

Fig. 30.9. Schematic of TMV assembly. A - The initiation region in the RNA forms a loop and enters the central channel of the protein disk. B - The disk transitions into the helical "lockwasher" form. C - New disks attach to the end of the RNA containing the loop. D - One of the RNA ends is continuously pulled through the central channel and interacts with new disks. E - Schematic representation of the RNA molecule in a partially assembled virus. The direction of RNA movement is indicated by the arrow

In addition to providing rapid nucleation, the double-layered disk significantly increases the Specificity of coat formation. A disk can bind to many nucleotides, whereas a single subunit binds to only three. Consequently, the disk exhibits a much higher selectivity for TMV RNA over host Cell mRNA than a single subunit does. Another important property of disks is that, under physiological conditions, they do not form helices in the absence of RNA. Two carboxyl groups in each subunit play a crucial role in this regard. At neutral pH, both carboxyl groups are ionized in the helical form, whereas only one is ionized in the disk. Electrostatic repulsion between closely spaced carboxylate ions in the helical form favors The formation of the disk. The binding of RNA to the helical form is accompanied by a sufficient Free energy change to overcome the electrostatic repulsion of the carboxylate ions. Thus, carboxylate ions act as a negative regulator that prevents helix formation in the absence of RNA.

30.4. Infection by Phage T4 Completely Reorganizes Macromolecular Synthesis in E. coli Cells

Bacteriophage T4 is a much more complex virus than TMV. Its double-stranded DNA contains approximately 165 genes compared to the 6 genes of TMV. However, the structure, reproduction, and assembly process of phage T4 have been studied quite thoroughly due to intensive genetic and biochemical analysis. The T4 virion consists of a HEAD, a tail, and six tail fibers (fibrils) (Fig. 30.10). Its DNA molecule is tightly packed inside an icosahedral protein shell, forming the virus head. The tail consists of two coaxial tubes connected to the head by a short collar. In the tail, a contractile sheath surrounds a central core through which DNA is injected into the host bacterium. The tail bears a baseplate at its end with six short spikes, from which six long, thin fibers extend.

Fig. 30.10. Electron micrograph of phage T4

The tips of the tail fibers bind to specific sites on the E. coli cell. As a result of ATP-dependent contraction, the sheath pulls the phage head toward the baseplate and tail fibers, causing the central core to penetrate The Cell wall, though not The Cell membrane. Then, the naked phage DNA crosses the cell membrane. Within a few minutes, all cellular DNA, RNA, and Protein Synthesis reactions halt, and the synthesis of viral macromolecules begins. In other words, the virus that has infected the cell takes over the synthetic machinery of the bacterial cell and replaces its genes with its own.

Table 30.2. T4 Phage Genes

The T4 phage DNA contains three groups of genes that are transcribed at different stages of infection: prereplicative (immediate-early), early, and late. Prereplicative and early Genes are transcribed and translated before T4 phage DNA Synthesis begins. Some of the Proteins encoded by these genes shut down the synthesis of host cell macromolecules. Shortly after infection, the host cell DNA is degraded by a deoxyribonuclease encoded by one of the early T4 phage genes. The T4 phage DNA itself is not hydrolyzed by this enzyme because it lacks clustered cytosine residues. Instead of cytosine, T4 phage DNA contains hydroxymethylcytosine (HMC). Furthermore, the HMC residues in T4 DNA are glucosylated.

These cytosine derivatives are incorporated into bacteriophage T4 DNA through the action of several phage-specific Enzymes synthesized during the Cytology/cytology/16.html">Early stages of infection. One of these enzymes hydrolyzes dCTP to form dCMP, preventing dCTP from being incorporated into T4 phage DNA. A second enzyme then introduces a hydroxymethyl group into dCMP, yielding 5-hydroxymethylcytidylate. A third enzyme converts 5-hydroxymethylcytidylate into the triphosphate, which serves as a substrate for DNA polymerases. Finally, a fourth enzyme glycosylates some of the hydroxymethylcytosine residues contained within the DNA.

The synthesis of late proteins is coupled with T4 phage METABOLISM/36.html">DNA Replication. At this stage, capsid proteins and Lysozyme are produced. Once progeny virion assembly is complete, lysozyme hydrolyzes and degrades the Introduction/37.html">Bacterial cell wall. Approximately 20 minutes post-infection, about two hundred new Viral Particles are released.