Paper : 03 Structure and Function of Biomolecules II Module: 08 Principal Investigator Paper Coordinator and Content Writer Content Reviewer Dr. Sunil Kumar Khare, Professor, Department of Chemistry, IIT-Delhi Dr. Sunil Kumar Khare, Professor, Department of Chemistry, IIT-Delhi & Prof. M.N. Gupta, Department of Biochemical Engineering and Biotechnology, IIT-Delhi Dr. Arun Goyal Professor, Department of Biotechnology Indian Institute of Technology Guwahati North Guwahati, 781 039 Assam, India 1
DESCRIPTION OF MODULE Subject Name Paper Name Module Name/Title 03 Structure and Function of Biomolecules II 08 Dr. Vijaya Khader Dr. MC Varadaraj 2
1. Objectives To know about viruses and their classification DNA and RNA viruses How do viruses infect and replicate in a host Virus-like particles like viriods and prions 2. Concept Map 3
3. Description 3.1 Introduction Viruses are undoubtedly one of the mysteries of the living world. This is mostly due to the fact that they are pseudo-living particles which show some chemical characteristics similar to living cells. The word virus is derived from the Latin word meaning poison; indeed many of them are contagious agents causing deadly diseases. Viruses have existed for thousands of years and have been responsible for outbreaks of smallpox, influenza, yellow fever and other pandemic diseases throughout history. By definition, viruses are submicroscopic acellular entities consisting of either DNA or RNA as their genetic material, often encapsulated inside a coat of protein. The virus particles are also called virions. They are obligate intracellular parasites that can only replicate and propagate within a suitable host cell. The viruses possess their own genetic elements, distinct and independent of the host genome, and can exist in extracellular form, outside the host cell for prolonged periods of time. Upon infection, viruses use the host metabolic machinery to carry out their replication cycle and produce more virus particles. They also use the host machinery for translation into mrna nad protein synthesis. The study of viruses is a significant part of microbiology and is specifically termed as Virology. The importance of viruses arises from the fact that in addition to being causal agents for the development of many diseases, they provide fascinating information regarding the biochemistry and genetics of 4
cellular processes. In recent times, scientists have also exploited viruses as remarkable tools in genetic engineering and biotechnology. 3.2 History of Viruses Since the ancient times, people have been acquainted with viral diseases, though the exact cause was unknown at that time. Viral epidemics such as smallpox and rabies often weakened native populations, leading to easier conquests by foreign invaders. Though it was known in earlier cultures, variolation or vaccination as a scientific technique of imposing protection to viral diseases by prior inoculation of attenuated viruses, was popularised by Edward Jenner in 1798. The first systematic studies regarding viruses were carried out on the Tobacco Mosaic Virus in the late 1800s. Dmitri Ivanovski showed that the sap of plants infected with TMV even after filtration to remove bacteria could induce infection in healthy plants, attributing this to the presence of a toxin. Later Martinus Beijerinck, through an independent study, proposed that the disease was caused by a filterable virus, an entity different from bacteria. By the 1930s, the TMV had been crystallised by Stanley and studies into its chemical nature by Bawden and Priries suggested viruses to be composed of proteins and nucleic acids. In 1931, Goodpasture grew the influenza and other viruses in fertilized chicken eggs, paving to the way for culture of viruses in vitro. The electron microscopic images of many viruses were obtained by the late 20 th century. More recently, scientists have explored the potential of viruses as tools for genetic engineering. 3.3 Structure of Viruses 5
Viral structures are diverse in their size, shape and chemical composition. The size of viruses vary between 30 to 400nm, making them difficult to study under light microscopes. Thus, the morphology of viruses has been extensively studied through various techniques such as electron microscopy, X-ray diffraction and biochemical or immunological analysis. The nucleic acid of a virion resides inside the particle surrounded by a protein shell known as capsid, which is composed of precise, repetitive assemblies of distinct proteins known as capsomeres. The complex entity of nucleic acid packaged inside the protein coat is termed as a nucleocapsid. One or more virus-specific enzymes are also present, which are involved in infection and replication. Nucleocapsids are mostly symmetrical in shape. 6
In general, there are four types of virion structure- 1. Icosahedral structures have 20 triangular faces and twelve vertices, appearing spherical under low power of microscopes. 2. Typical viruses with helical symmetry, such as TMV, are shaped like hollow protein cylinders. 3. Enveloped viruses have an outer membrane over their nucleocapsid, giving them a roughly spherical shape, though inside the nucleocapsid may be spherical or helical. The envelopes are mostly composed of lipids. 4. Complex viruses are neither icosahedral nor helical. They can have different parts, each having separate shapes and symmetries. Some of them have tails or multi-layered walls around their nucleic acid. Bacteriophages fall under this category. 3.4 Virus Classification The process of naming viruses and placing them into taxonomic units is termed as virus classification. The definition of species, form the basis of any biological classification system. Due to their pseudoliving nature, it is often difficult to define a species of virus and categorise them according to established classification systems for other cellular organisms. Since 2013, the International Committee on Taxonomy of Viruses (ICTV) defines a species of virus as- "A species is a monophyletic group of viruses whose properties can be distinguished from those of other species by multiple criteria." 7
The main characteristics used for virus classification are phenotypic characteristics, such as morphology(capsid symmetry and presence of envelope), characteristics of nucleic acid present, mode of replication, host organisms, and the type of disease they cause. The two main schemes used for the classification of viruses are- ICTV system and Baltimore Classification system The ICTV system uses the Phenotypic characteristics, while the Baltimore classification differentiates according to the type of nucleic acids present and their mode of replication inside a host. 3.2.1 ICTV classification system This classification system started in the 1970s, and it is still being modified. It features a similar taxon structure as used in the classification of cellular organisms. This classification starts at the level of order and continues as follows: Order (-virales) Family (-viridae) Subfamily (-virinae) Genus (-virus) 8
Species Species names generally take the form of the name of the disease the virus is responsible for, such as [Disease] virus. Currently (2012), according to the ICTV, seven orders, 96 families, 22 subfamilies, 420 genera, and 2,618 species of viruses have been defined. 3.2.2 Baltimore classification system The Baltimore system was formulated first in 1971 and uses the nucleic acid present in viruses as the basis of classification. In this system, the type of nucleic acid present(dna or RNA), strandedness(single or double stranded), sense(positive or negative) and mode of replication are taken into consideration for classification. Viruses are actually remarkable in using all types of nucleic acids as their genetic material, that is, single stranded DNA, double stranded DNA, single stranded RNA and double stranded RNA. This gives them enough flexibility. Animal viruses are found to contain all four types of nucleic acids, while plant viruses harbour RNA as their genetic material. Phages mostly have single stranded DNA or RNA, while bacterial viruses contain double stranded DNA. Many a time different viruses may cause the same disease. In addition, many viruses possess similar structures which are very difficult to determine, even under microscope. Thus, the classification of viruses based on their genome overcomes such inadequacy as those belonging to the same group will behave in a similar fashion. Thus, according to the Baltimore system of classification, viruses are segregated into seven of the following classes- 9
DNA Viruses- 1. dsdna- The first group of Baltimore classification has viruses which have double stranded DNA as their genome and replicate via a DNA-dependent DNA polymerase. They can be both circular or linear. Some families have circularly permuted linear genomes while others have linear genomes with covalently closed ends. Pox, herpes viruses and many phage viruses come under this category. 2. ssdna- Single-stranded DNA viruses belong to Group II of the classification system, and include viruses which are found in sea water, fresh water, sediment, terrestrial, extreme, metazoan-associated and marine microbial mats. Microviridae is a major family of this type. These viruses create a doublestranded DNA intermediate form using the host s DNA polymerase. The number of ORFs range from one to eight. RNA Viruses- 3. dsrna- Group III includes the most diverse group of viruses, including rotaviruses which cause gastroenteritis in young children. Evolutionarily, dsrna are generally destroyed on contact with enzymes such as Dicer. Thus they could be developed as antiviral agents against these viruses. 4. ssrna Plus strand- The IV group of Baltimore classification has positive, sense-strand RNA as the genetic material, which can act as messenger RNA to translate into protein. The replication occurs via double stranded RNA intermediates. Positive-sense ssrna virus genomes contain RNA-dependent 10
RNA polymerase, a viral protein that synthesizes RNA from an RNA template. Many pathogenic viruses such as SARS, MERS, dengue virus, hepatitis C belong to this category. 5. ssrna Minus strand- This viruses of group V have negative, sense-strand RNA, which the complementary to the messenger RNA, thus they are not infectious by themselves. They also need a RNA-dependent RNA polymerase to form the mrna which then translates to capsid proteins. Viruses of families Bornaviridae, Arenaviridae, Deltavirus and Varicosavirus fall under this category. Reverse transcribing Viruses- 6. RNA to DNA hybrid- Group VI is of the very well-known retroviruses and include HIV. The virions contain a RNA dimer as the genome. Instead of direct translation, these viruses first convert their RNA into DNA by Reverse Transcriptase and integrate it with the host genome using the enzyme Integrase. If this integration occurs in the germ cells of the host, they are transmitted to the next generation. The RNA synthesized enters the cytosol to produce different proteins, such as the gag gene into molecules of the capsid protein, the pol gene into reverse transcriptase, and the env gene is translated into molecules of the envelope protein. Retroviruses such as Rous sarcoma virus and Mouse mammary tumor virus are known to cause cancers. 7. DNA to RNA hybrid- Also known as pararetroviruses, they are not considered true DNA viruses, and replicate via a RNA intermediate. Hepadnaviridae and Caulimoviridae are included in this class. Some differences between DNA and RNA viruses are as follows- 11
DNA Viruses RNA viruses Double stranded DNA viruses are more common Single stranded RNA viruses are more common They replicate inside the nucleus of the cell They replicate in the cytoplasm DNA viruses integrate with the host genome and use its machinery to synthesize proteins. Thus they modify the host genome. RNA viruses inject their RNA into the cytoplasm of the host, and synthesize protein. Host DNA is not modified. First transcription to RNA occurs, followed by translation to protein The transcription stage can be skipped and direct synthesis of protein occurs. Mutation levels are low in DNA viruses, as DNA polymerase has proofreading activity RNA viruses mutate quite fast, as polymerase is error prone RNA 12
Viral replication All viruses, as obligate intracellular parasites must attach, enter and use the cellular machinery and enzymes of their host for their reproduction. During the late phase of infection viruses steer the host s machinery to synthesize large amounts of viral mrna and proteins rather than the cell s normal macromolecules, which are then assembled into complete viral particles and released. The stages of replication can be studied using infected cultured cells and doing infectivity assay at regular intervals 13
of time. The viral replication is often represented by a One-step growth curve, where time is shown in the x-axis and extracellular virus levels on the y-axis. At first, during attachment and penetration, the levels of extracellular viruses decrease drastically during the eclipse phase. During the logarithmic/expansion phase, the start of the virus assembly happens, the cells are lysed and new virions are released. As the effects of the viral replication show, the host cell eventually die, causing viral population to plateau off. The typical time required to reach the death phase may vary from 60 minutes in bacteriophages to more than 24 hours in animal viruses. The typical features of viral replication are- 1. Attachment- This step involves absorption of the virion to the susceptible host. The host specificity of a particular virus depends much on its attachment process, which is mediated by the interaction between viral attachment proteins present on the envelope or inside the capsid and specific cell surface components called receptors on the host cells. These receptors are generally surface molecules on the plasma membrane to carry out normal functions of the cell, such as proteins, carbohydrates, lipids or their complexes. Thus, in the absence of a particular receptor or mutations in an existing one, renders the virus unable to attach and gives resistance to the host cell. However, many animal viruses are able to attach to more than one receptor, so the loss may not prevent attachment. Different tissues in a multicellular organism express different receptors, thus animal viruses often affect only a particular tissue. 14
2. Penetration- Attachment is closely followed by penetration where the virus particle is uncoated, i.e releases its genetic material from the capsid to inside of the cell. In other animal viruses, the capsid is engulfed by the host plasma membrane by an invagination and a vacuole containing the capsid enters the cell. It is uncoated inside, alterations in the viral structure create a pore, from which the genetic material is released into the cytoplasm. Now the genetic material is needed to be read by the host machinery, otherwise infection is fruitless. A host cell which allows complete replication cycle is said to be permissive for that virus. To counteract viruses, host cells often possess restriction endonucleases which destroy viral DNA. 3. Synthesis- During this step, the virus directs the host machinery for the synthesis of nucleic acid and protein. The proteins then form the coat, capsid or accessory proteins. A number of Early and Late genes are expressed to synthesize proteins to help carry the processes before and after replication. The difference between DNA and RNA viruses exists mainly due to the process in which they replicate within the host cell. Double stranded DNA viruses enter into the nucleus of the cell, and integrate their DNA within the host genome. The host polymerase then replicates this DNA, and transcribes it into mrna, which then travels to the cytoplasm, to synthesize the proteins using the host RNA polymerase. Many double stranded viruses, also contain polymerases, thus they directly enter the cytoplasm. For many RNA viruses, a special enzyme called the Reverse Transcriptase is employed, which converts the viral RNA into DNA which integrates into the host genome, then transcribing into the 15
mrna of necessary proteins followed by transcription. Other RNA viruses, skip this reverse transcription and directly translate to protein using the host s machinery. 4. Assembly and Maturation- The synthesized capsids and genomes are channelled to different locations in the cell(nucleus or cytoplasm) for assembly and release. The capsids may be either built around the genome or packed during the final maturation step. These assemble as units of structural proteins called as capsomers. For example for poliovirus, the structural proteins first associate as trimers, then bind to form pentamers, and twelve such pentamers form the icosahedral capsid structure, to which the genomic RNA is sequestered. For simple viruses such as HPV, two major proteins form the pentamers which proceed to develop the icosahedron. During genome packaging, long concatamers of genomic DNA are excised at specific points by restriction endonucleases, and these individual molecules enter the capsid through an open corner which is further sealed. For enveloped viruses membrane components are assembled and packed with viral genomes. The whole process is termed as maturation. 5. Release- The assembled particles first bud out in an enveloped form from the nuclear compartment and then fuse with the outer cell membrane. The release of the assembled virus particles may occur by budding through the cell membrane or by cell-lysis releasing the progeny viruses. For viruses such as influenza and HIV, recognition of specific packaging sequences occur followed by the release of the particles by budding at the plasma membrane of the host cell. Small untranslated regions of the RNA 16
molecules often help in selective packaging to ensure one copy of each viral segment is packed before budding. Some viruses such as the herpesvirus may also infect an adjacent cell by cell-cell fusion. The lytic and lysogenic cycle Broadly, the life cycle of a bacteria consists of a lysogenic and a lytic cycle. In the lysogenic cycle, the virus injects its genetic material into the host, and is followed by integration of the viral DNA into the host DNA. The virus then uses the host machinery to replicate into number of copies. The virus can rest in this cycle for some time without killing the host cell. From the lysogenic cycle, the virus can proceed to the lytic cycle inside the host cell. In the lytic cycle, the capsids are produced, the virus particles assemble, and finally the cell is ruptured to release the mature virus particles. 17
Viroids Viroids are sub- viral pathogens, mostly infecting plants, consisting of a circular, non-coding singlestranded RNA, without protein coats. Their genomes are much smaller than that of a virus, consisting of 200-500 nucleotides as compared to that of viruses, which have a genome size of around 2000 nucleotides. Viroids use the host s replication mechanism, using the RNA polymerase II, which catalyses the rolling circle synthesis of RNA from the viroid DNA as template. Prions Prions are an extraordinary class of infectious agents, quite different from viroids. They are entire made up of protein, and lack DNA or RNA. Prions are the causative agents of a number of animal diseases such as scrapie in sheep and Bovine spongiform encephalopathy(bse) or mad cow disease and chronic diseases in human being such as kuru and Creutzfelt Jacob disease. So, how do prions infect in the absence of nucleic acids? Research has found that the host cell contains a gene named PrnP coding for a protein PrP c found mostly in the neurons of the brain. The pathogenic form of this protein, designated as PrP Sc, is conformationally different(containing more α-helical segments) from the wild-type protein(containing more β-sheet regions). When a pathogenic protein enters a host cell, it converts PrP c to PrP Sc resulting in the disease condition. This protein is insoluble and aggregates in the brain, thus causing its destruction. Normal prions act as cytoplasmic membrane glycoprotein and membrane attachment is essential for the pathogenicity. Thus, prion protein replicates itself by inducing conformational changes in normal wild-type prion proteins. 18
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