high-resolution images that reveal the structure of assembled phage particles. Our goal is to create a detailed picture of the forces implicated in DNA packaging and ejection and, most importantly, make quantitative predictions that can be tested experimentally.
Figure 3.1: Life cycle of a bacterial virus. The ejection of the genome into the host cell happens within a minute for phage like λ and T4 [14, 40]. The eclipse period (time between the viral adsorption and the first appearance of the progeny) is about 10-15 minutes [41]. The packaging of the genome into a single capsid takes about 5 minutes [11]. Lysis of the bacterial cell is completed in less than an hour [41].
The chapter is organized as follows: In Section 5.1 we provide a brief introduction to viruses in general, and bacteriophages in particular. In Section 3.2 we examine dsDNA bacteriophage with the aim of assembling the relevant insights needed to formulate quantitative models of packing and ejection. In Section 3.3 we develop the model by examining the DNA packaging process in detail, and in Section 3.4 we show that it can be used to explain the DNA ejection process as well. In Section 3.5 we take stock of a range of quantitative predictions that can be made on the basis of the model and suggests new experiments.
Despite their evident malice, historically the study of virus has served as both an inspiration and testing ground for the basic theories of genetics and structural biology and has been an object of affection for researchers from various fields. Recently, viruses have resumed their role as a testing ground for quantitative modeling in biology, and a part of the aim of the present thesis is to apply mechanical modeling to the processes involved in the forementioned assault on the host cell.
3.1.1 Major Groups of Viruses
It is customary to subdivide viruses according to the nature of their hosts into plant viruses, animal viruses and bacterial viruses or bacteriophages (bacteria eater) [10]. Even such broad divisions can create ambiguities with some plant viruses that can multiply in their insect vectors. Each virus has a range of hosts in which it can reproduce. Viruses are discovered as pathogens, and hence it is logical to classify them in terms of their major host, the host whose response to the viruses first came to the attention of man.
1: Animal viruses
The only invertebrates in which virus diseases have been reported are the insects, which are eco- nomically the most important and therefore most thoroughly studied class of invertebrates.
Virus diseases are known in fish and in amphibia. In birds one finds many virus diseases, some of them economically very important, like Newcastle disease and laryngotracheitis. Virus diseases have been recognised in most domestic mammals as well as in many wild ones. Virus diseases of humans include such major epidemiological problems as smallpox, yellow fever, mumps, rabies, AIDS, SARS, etc.
2: Plant viruses
There are relatively few known viruses in gymnosperms, ferns, fungi or algae. The flowering plants on the other hand, are hosts to many types of viruses. The viruses rank next to fungi in causing plant diseases of economic importance including diseases of potatoes, beans, beets, tobacco, sugarcane, cocoa, and fruit crops. Historically the importance of plant viruses lie in the fact that the very existence of viruses was discovered by Dutch scientist Martinus Beijerinck while working on the tobacco mosaic disease, which we now say to be caused by the Tobacco Mosaic Virus (TMV) [9].
3: Bacterial viruses or bacteriophages
Bacteriophage literally means bacteria eater. There is hardly a group of bacteria for which bacte- riophages (phage for short) have not yet been discovered. Bacteria for which no phage has yet been reported are those about which our knowledge is quite limited. The phage specificity in attacking the host may be narrower or broader then the classification boundaries that separate the bacterial genera and species. For example, a phage may multiply only on a certain strain ofEscherichia coli, whereas another phage reproduces in many strains of E. coli and the closely allied genusShigella.
Since bacteria can acquire phage resistance by discrete mutational steps, a strain sensitive to several
phages may produce a series of stable mutants resistant to one or more of the phages.
The phage is the simplest viral system. The phage life-cycle is measured in minutes, whereas the infectious cycles of plant and animal viruses occupy hours or days. Phage replicate in bacterial hosts that can be readily obtained by an overnight culture, contrary to the plant or animal cells, which take much longer to grow to concentrations sufficient for infection. Due to these reasons, bacteriophages provide a tractable experimental system for analysis and have expedited the progress in virology in the past and the present [9]. It is precisely because of this simplicity of the phage and the vast experimental literature on it that we shall refer to phage as the model system for our mechanical modeling.
Bacteriophage have served as a historical centerpiece in the development of molecular biology.
For example, the classic Hershey-Chase experiment [42, 43] that established nucleic acid to be the carrier of the genetic blueprint was performed using bacteriophage T2. The biology of bacteriophage λprovided a fertile ground for the development of the understanding of gene regulation [44], while the study of virulent T phages (T1 to T7) paved the way for many other advances such as the definition of a gene, the discovery of mRNA, and elucidation of the triplet code by genetic analysis [43]. One of the central themes of the present chapter is that phage are similarly poised to serve as compelling model systems for quantitative analyses of biological systems.
Henceforth we will mostly discuss phages, and if the word virus is used without any additional qualification, it should be taken to mean phage.
3.1.2 Structure of a Typical Bacteriophage
The phage particles are elegant assemblies of viral, and occasionally cellular, macromolecules. They are marvellous examples of architecture on the molecular scale with form beautifully adapted to the functions of the phage. The structure of a typical phage is as shown in Fig. 3.2
1: Genetic Material
The phage genome carries the entire blueprint for its propogation. The phage genome can be one of the following nucleic acids: single-stranded (ss) DNA, double-stranded(ds) DNA, ssRNA, or dsRNA.
There is a flurry of diabolic activities once the viral genome gets inside the host cell. The genetic code on the genome is such that the cellular machinery is tricked into synthesizing the enzyme RNA polymerase for the virus, and after that the virus essentially takes over the host cell. The RNA polymerase produces mRNA, which in turn produces all the proteins encoded in the genetic code and makes copies of the genome. The proteins and the genome thus synthesized then assemble to form new viruses, all at the expense of the cellular resources. The host cell breaks open after this attrition and the new virus particles are liberated. The nucleic acid dsDNA being the human genetic material is studied the most among the above mentioned nucleic acids. As a result, its chemical,
structural and, more importantly for us, mechanical properties are better known (explained in Section 3.6.1). We will, hence, investigate only the dsDNA phages.
Neck (connector)
Figure 3.2: Structure of a typical bacteriophage. This is bacteriophage T4 [39]. It has a capsid, neck, tail, base-plate, and tail-fibers. These components are composed of proteins synthesized by the virus inside the host cell. The genetic material is protected inside the capsid.
2: Capsid (Head) and neck
The genetic material is enclosed inside the capsid, which is made up of protein subunits assembled according to relatively simple geometrical principles based on elementary physical considerations.
There are only two basic types of capsids, helical and isometric (or quasi-spherical) [10]. Each of these arrangements is reached by the capsid proteins by a process of self-assembly that must be energetically favorable. As can be seen from Fig. 3.3a, the recent advancements in cryoelectron microscopy (CEM) have made it possible to image the structure of viral capsids in amazing detail.
The main function of the capsid is to protect the genetic material from large variations in the temperature, pH, or chemical composition of the environment. The DNA is organized in the capsid in a very regular fashion (see Fig. 3.3b).
3: Neck or Connector
The connector is the junction between the head and the tail of the virion and is the point of DNA entry during packaging and exit during the ejection. The connector forms a part of the portal motor that utilizes ATP hydrolyses to drive DNA translocation and package it inside the phage capsid [11].
5: Tail, baseplate and tail fibers
Many but not all phages have tails attached to the phage head. The tail is a hollow tube through which the nucleic acid passes during infection. The size of the tail can vary, and some phages do not even have a tail structure. In the more complex phages like T4 the tail is surrounded by a
a) b)
Figure 3.3: The structure of viral capsid and the organisation of the DNA inside. (a) The viral capsid can be either isometric (or quasi-spherical) or organized in a helical fashion (figure taken from Baker et al. [38]). (b) The cryoelectron micrograph (CEM) of the genome inside the capsid of bacteriophage T7. The DNA is arranged in a very regular fashion (figure taken from Cerritelli et al. [17]).
contractile sheath which contracts during infection of the bacterium. At the end of the tail the more complex phages like T4 have a base plate with one or more tail fibers attached to it. The base plate and tail fibers are involved in the adsorption of the phage to the bacterial cell. Not all phages have base plates and tail fibers. In these instances other structures are involved in binding of the phage particle to the bacterium. The tail fibres are used to hold on the outer layer of the host cell while the virus is preparing to launch its genome inside the host-cell.