BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 30. VIRUSES

30.11. Poliovirus Proteins Are Produced by Multiple Cleavage of a Giant Precursor

Poliovirus consists of a single-stranded (+) RNA 7.5 kb in length, enclosed within an icosahedral capsid. Upon entering the Cytoplasm of the host Cell, this virion RNA molecule serves as a template for Protein Synthesis. It is translated by the host cell Ribosomes to produce capsid Proteins and a specialized RNA-dependent RNA polymerase (RNA replicase). Subsequently, the RNA replicase synthesizes (-) strands using the viral (+) RNA as a template (Fig. 30.21). The (-) RNA, in turn, serves as a template for the synthesis of numerous (+) strands, which participate in protein synthesis or become packaged into capsids to yield new virions.

Class="center">Fig. 30.20. Electron micrograph of poliovirus particles

Fig. 30.21. Replication of poliovirus RNA

A striking feature of poliovirus Gene Expression is that the virion (+) RNA serves as a template for the synthesis of a continuous polypeptide chain containing more than 2,000 amino acid residues. David Baltimore showed that this giant polypeptide is cleaved by host cell proteases into seven proteins: four coat proteins, one RNA replicase, and two proteins whose Functions remain unknown (Fig. 30.22). The newly synthesized polypeptide chain is cleaved into three pieces, which then undergo further Cleavage. Notably, two of the coat proteins are generated from a precursor at The final stage of virion assembly.

Fig. 30.22. Synthesis of poliovirus proteins via multiple cleavage of a giant polypeptide precursor

Why does poliovirus synthesize its proteins in such a seemingly complex manner? Evidently, it has no choice. Recall that in Eukaryotic Cells, an mRNA molecule can, for reasons not yet fully understood, yield only a single polypeptide chain upon Translation. Prokaryotic mRNAs, by contrast, are frequently polycistronic (e.g., the mRNA of the lactose Operon). Thus, poliovirus utilizes polyprotein cleavage to overcome the constraints imposed by the Features of the animal host cell.

30.12. The Genomic RNA of Vesicular Stomatitis Virus Is Transcribed into Five Monocistronic mRNAs

Vesicular stomatitis virus, which causes a mild disease in cattle, and rabies virus represent the second mode of gene expression. Their virions contain a single-stranded (-) RNA molecule that cannot function as a template. Therefore, the first step in its expression is the synthesis of (+) RNA. Since uninfected cells lack RNA replicase, the Viruses must carry this enzyme within the virion and inject it into The Cell upon infection. Indeed, two of the five VSV virion proteins mediate RNA replication. The infectiousness of the virus requires the large L protein (200 kDa) and the nonstructural NS protein (45 kDa), which are present in small amounts. The genomic RNA is complexed with numerous molecules of the nucleocapsid N protein (50 kDa), the major virion protein. VSV is enclosed in a Lipid Bilayer Membrane acquired from The Plasma Membrane during the budding process (Fig. 30.23). The virus-encoded G protein (glycoprotein, 65 kDa) forms spikes protruding from this membrane. The matrix M protein (29 kDa) is located between the envelope and the nucleocapsid. These five VSV proteins are produced by the translation of five (+) mRNAs rather than by the cleavage of a single polyprotein. The same RNA replicase also synthesizes a long (+) RNA containing all the Genetic information of the virus. This full-length (+) RNA, in turn, serves as a template for the synthesis of (-) RNA, which is packaged to yield new virions.

Fig. 30.23. Electron micrograph of vesicular stomatitis virus

Fig. 30.24. Electron micrograph of reovirus

Rhabdoviruses are bullet-shaped viruses. They include the vesicular stomatitis and rabies viruses. The name derives from the Greek word rhabdo, meaning "rod".

30.13. The Reovirus Genome Consists of Ten Distinct Double-Stranded RNA Molecules

Reovirus, which contains double-stranded RNA, infects mammalian cells and represents the third type of viral genetic system. The virion core contains ten different double-stranded (±) RNA molecules associated with proteins. Upon entering the host cell, the virion sheds its outer icosahedral shell, which is composed of Three types of proteins. Removal of this coat activates the RNA polymerase contained within the virion core. This RNA-dependent polymerase fully transcribes the 10 (+) RNA molecules, so that the resulting (+) mRNAs are identical in length to The Genome fragments. The (±) RNA template is transcribed asymmetrically and conservatively—that is, only (+) RNAs are synthesized, and the parental (±) RNAs are degraded. Within the virion core, caps are added to the 5' ends of these mRNAs by enzymatic action. These ends then emerge through channels in the core (Fig. 30.25). Consequently, the core is a highly organized system for mRNA synthesis. Each of these ten mRNAs yields a single protein upon translation. Subsequently, the entire set of ten (+) RNAs associates with certain viral proteins to form a core precursor (precore), in which the ten (-) strands are synthesized.

Fig. 30.25. mRNA synthesis within the reovirus core. The mRNA molecules appear as filaments extending from the dark core bodies

Reovirus — a double-stranded RNA virus isolated from the respiratory and gastrointestinal tracts of humans and other mammals; as far as is known, it does not cause disease. The prefix reo is formed from the first letters of the English words respiratory enteric orphan, meaning an enteric-respiratory orphan virus (an orphan because it wanders around “unattached” to any disease).

Why is the reovirus genome segmented? As noted above, animal viruses cannot have polycistronic mRNAs. Poliovirus solves this problem by cleaving a giant precursor protein, whereas vesicular stomatitis virus transcribes virion RNA into short mRNAs, each corresponding to a single protein. The reovirus strategy is to have a separate “chromosome” for each synthesized protein.

The fourth pathway for expressing the genetic information of Introduction/7.html">RNA-containing Viruses is The Use of a DNA intermediary that integrates with the host cell genome. This is a more complex genetic system utilized by Retroviruses (RNA-containing tumor viruses). We will discuss it later in this chapter (Sec. 30.19).

30.14. Small RNA-Containing Phages Contain Overlapping Genes

RNA-containing phages such as R17 (MS2, F2) and Q_e belong to the simplest viruses. They have a regular polyhedral shape and a diameter of about 200 А. The capsid of these closely related phages contains 180 molecules of coat protein with a mass of 14 kDa and one molecule of protein A (maturation protein) with a mass of 38 kDa. In addition, the single-stranded (+) RNA encodes one of the replicase subunits. Until recently, it was believed that these small RNA-containing viruses contained only three genes. However, the discovery of a phage mutant that forms normal virions but is incapable of lysing the host cell prompted the search for yet another virus-encoded protein. Indeed, RNA-containing phages possess a fourth gene that encodes a protein required for lysis of the host bacterium. The gene for this lysis protein overlaps with the genes for the coat protein and the replicase subunit (Fig. 30.26). These small RNA-containing phages, much like the small DNA-containing phage ɸX174 (Sec. 26.11), use overlapping genes to pack more information into their small genomes. The virion (+) RNA molecule serves as a template for the synthesis of both four proteins and (-) RNA. Subsequently, the (-) RNA is used as a template to produce multiple copies of (+) RNA. Thus, in terms of their genetic system, these phages resemble poliovirus.

Fig. 30.26. Overlapping genes in the RNAs of phages R17 and Qβ. One reading frame is shown in yellow, and the other in blue

The replicase that synthesizes the (+) and (-) chains of phage RNA is a remarkably interesting enzyme. It exhibits high Specificity for homologous phage RNA. Consequently, host cell RNA molecules do not compete with phage RNA during replication. Qβ replicase consists of four subunits, of which only one is encoded by phage RNA. The other three replicase subunits are host cell proteins that the phage has co-opted for its own needs. Two of them are the protein synthesis elongation factors EF-Tu and EF-Ts, and the third is a component of the 30S ribosomal subunit. In this manner, phage Qβ constructs a highly specific enzyme in the most economical way possible.

Regulatory mechanisms ensure the correct temporal sequence of translation and replication. (+) RNA serves simultaneously as a template for PROTEIN SYNTHESIS AND for (-) RNA Synthesis. It would be undesirable for both processes to occur simultaneously on the same (+) RNA molecule, as ribosomes moving in the 5' → 3' direction would collide with the replicase moving in the 3' → 5' direction. This does not happen because Qβ replicase strongly inhibits ribosome binding to the (+) RNA until a sufficient number of (-) RNA molecules have been synthesized.

The four phage proteins are synthesized in varying amounts. The coat protein, which is synthesized throughout the entire infection period, is the major product of translation. One reason for this is that ribosomes bind much more tightly to the initiation site of the corresponding Cistron than to other initiation sites in the (+) RNA. In addition, the coat protein represses Translation of the replicase gene by blocking its initiation site. Thus, the coat protein acts as a specific translational repressor. Protein A is translated only from incomplete (+) RNA molecules because in full-length RNA molecules its initiation site is blocked As a result of base pairing. The Secondary Structure of the full-length RNA molecule permits the translation of only a small amount of protein A.