BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 30. VIRUSES

30.8. The Flexibility of the TMV Coat Protein Enables It to Form an Icosahedral Capsid

Tomato bushy stunt virus (TBSV) is a spherical virus that illustrates another principle of viral Organization (Fig. 30.15). TBSV contains a single RNA molecule, 4800 NUCLEOTIDES in length, enclosed within a shell composed of 180 identical protein subunits with a mass of 41 kDa. How are these coat subunits arranged? The maximum possible Symmetry for a closed shell is achieved by an icosahedron, which has an order of 60 (Fig. 30.16, A). In other words, no more than 60 identical subunits can be packed into a spherical shell while maintaining absolute symmetry. However, TBSV and several other spherical Viruses contain 180 identical subunits. The biological advantage of constructing a shell from 180 subunits instead of 60 of the same size is that a larger virion can package more nucleic acid. This is accomplished not by breaking symmetry, but by relaxing it somewhat (Fig. 30.16, B).

Class="center">Fig. 30.15. Electron micrograph of tomato bushy stunt virus

Fig. 30.16. Icosahedral surface lattice demonstrating the packing of 60 identical subunits (A) and 180 quasi-equivalent subunits (B). Note that all tail-to-tail contacts in Fig. A are formed by ring-like groups of five subunits, whereas in Fig. B some of these contacts are formed by groups of five, and others by groups of six subunits

High-resolution X-ray crystallographic analysis of TBSV, conducted by Stephen Harrison, revealed that the chemically identical subunits of its coat can be divided into three groups (A, B, and C), each consisting of 60 Proteins that obey strict icosahedral symmetry. At the same time, molecules from different groups are positioned somewhat differently relative to one another; hence, they are called quasi-equivalent.

How can quasi-equivalence be explained from a physical standpoint? X-ray analysis showed that each subunit consists of an S-domain, which forms part of the shell surface; a P-domain, which projects outward; and an N-terminal segment directed inward. The P- and S-domains of all subunits have approximately the same Structure. However, the angle between the P- and S-domains in group C subunits differs markedly from that in subunits A and B. The P- and S-domains are connected by a hinge that allows a rotation of up to 20°. Another difference is that the N-terminal region is ordered in group C subunits, whereas it is in a disordered conformation in subunits A and B. Because of this structural flexibility, subunits belonging to different groups can interact with one another in an almost identical manner, which is essential for self-assembly. On the other hand, the RNA inside the particle does not appear to have a definite conformation. The flexible N-terminal segments of the coat subunits penetrate inward and interact with the RNA.

30.9. Bacterial Restriction Endonucleases Cleave Foreign DNA Molecules

We have already seen that phage T4 possesses an enzymatic system for the Selective Cleavage of host Cell DNA. Similarly, Bacteria possess Enzymes known as Restriction Endonucleases, which cleave foreign DNA molecules. These enzymes were discovered through the observation that phages grown on one bacterial strain (e.g., E. coli B) grow poorly on another strain (e.g., E. coli K), and vice versa. This phenomenon, whereby a phage grows poorly on a strain different from the one on which it was propagated, was termed restriction. However, a small fraction of the phage (approximately 10^-5) escapes restriction and subsequently grows well in the new host. At the same time, these phages lose The ability to grow on the old host.

All these findings indicated that specific modification within the host Cells protects the phage from restriction. Werner Arber then demonstrated that specific host-cell modification actually acts upon the phage DNA and that restriction results from the degradation of phage DNA. Host cell DNA and other DNA molecules contained within host cells are methylated at specific sites. These same sites are recognized by the restriction endonuclease, which cleaves only unmethylated sequences. Thus, methylation of a specific base in the target sequence (recognition site) prevents Hydrolysis by the restriction enzyme (Fig. 30.17). The timing of this modification is critically important: Bacterial DNA is not cleaved because it is methylated beforehand. A newly replicated bacterial chromosome, methylated on only one (parental) strand, is resistant to the action of the restriction enzyme. Such hemimodified DNA becomes fully methylated before THE START OF the next Replication cycle.

Fig. 30.17. Methylation of recognition sites (target sequences) protects them from cleavage by restriction endonucleases

Two Types of restriction-modification systems have been found in bacteria. In type I systems, methylase and nuclease activities are associated with a large, multi-subunit complex. For instance, such complexes in E. coli K and B consist of Three types of polypeptide chains. The α-chain possesses endonuclease activity, the ß-chain possesses methylase activity, and the γ-chain bears the DNA recognition site. Type I enzymes require S-adenosylmethionine and ATP for both nuclease and methylase activities. Type I enzymes cleave unmodified DNA at random sites 1900 Base Pairs or more 5' to the recognition site¹ while simultaneously hydrolyzing ATP. In type II systems, by contrast, methylases and Nucleases are separate. S-adenosylmethionine serves as the methyl group donor in the modification reaction, but is not involved in DNA cleavage. Another difference is that type II nucleases and methylases do not require ATP. Most remarkably, the cleavage sites of type II nucleases are highly specific. As discussed in a previous chapter (Section 24.27), many of these enzymes recognize a specific sequence of 4 to 6 base pairs and hydrolyze a single, strictly defined phosphodiester bond within each strand in that region. A distinctive feature of these cleavage sites is that they possess 2-fold rotational symmetry (Fig. 30.18). Restriction enzymes are an indispensable tool in The Study of Introduction/20.html">DNA Structure (Section 24.27) and the construction of novel DNA molecules (Section 31.9).

¹ Arber studied the type I restriction-modification system using the single-stranded DNA phage fd, which allowed him to specify THE POSITION OF the cleavage site relative to the recognition site as shown in the text. — Transl. note.

Fig. 30.18. Specificity of the restriction endonuclease Hind III from Hemophilus influenzae and its corresponding methylase. The 2-fold axis of symmetry is shown in green

30.10. Replication Strategies of RNA Viruses

The replication of RNA viruses presents a unique challenge because uninfected host cells lack enzymes for synthesizing RNA from an RNA template. Consequently, RNA viruses must carry the Genetic information for synthesizing an RNA-dependent RNA polymerase (also called RNA replicase or RNA synthetase) or an RNA-dependent DNA polymerase (also called Reverse Transcriptase). RNA viruses are conveniently classified According to the type of RNA contained within the virion and their mRNA. By definition, mRNA is (+) RNA, and the strand complementary to it is (-) RNA. Four pathways of RNA virus replication and METABOLISM/31.html">Transcription are known (Fig. 30.19). Class 1 viruses (e.g., poliovirus) contain a positive-strand RNA. They synthesize (-) RNA, which serves as a template for The production of (+) mRNA. Class 2 viruses (e.g., rabies virus) contain a negative-strand RNA, in which the virion (-) RNA serves as a template for the synthesis of (+) mRNA. Class 3 viruses (e.g., reoviruses) contain double-stranded RNA; the virion (±) RNA directs the asymmetric synthesis of (+) mRNA. The most unusual are class 4 viruses, the Retroviruses (e.g., Rous Sarcoma virus). In these viruses, the expression of genetic information contained in the virion (+) RNA is mediated by The formation of DNA, which then serves as a template for the synthesis of (+) RNA. Thus, the flow of genetic information in retroviruses goes from RNA to DNA and then back to RNA.

Fig. 30.19. Modes of Gene Expression IN RNA viruses

Picornaviruses are a group of small (pica) RNA-containing (RNA from English) viruses. A single-stranded (+) RNA molecule is surrounded by an icosahedral protein coat with a diameter of 270 Å. Picornaviruses include the poliovirus, rhinovirus (which causes the common cold), and FOOT-and-Mouth disease virus in cattle.