Viruses: Study of Viruses PDF

1  STUDY OF VIRUSES Viruses are simple, acellular entities. Most possess only one type of nucleic acid, either DNA or RNA and they reproduce only within living cells because they are obligate intracellular parasites. Early Development of Virology Although the ancients did...

1  STUDY OF VIRUSES Viruses are simple, acellular entities. Most possess only one type of nucleic acid, either DNA or RNA and they reproduce only within living cells because they are obligate intracellular parasites. Early Development of Virology Although the ancients did not understand the nature of their illnesses, they were acquainted with diseases, such as rabies, that are now known to be viral in origin. Great epidemics of A.D. 165 - 180 and A.D. 251 - 266, weakened the Roman Empire, may have been caused by measles and smallpox viruses. In the 18th century an English country doctor, Edward Jenner, began inoculating humans with material from cowpox lesions. Although Jenner did not understand the nature of smallpox, he did manage to successfully protect his patients from the dreaded smallpox disease through exposure to the cowpox virus. He published the results of 23 successful vaccinations in 1798. 2 Until 19th century, harmful agents were often grouped together and sometimes called viruses [Latin virus, poison or venom]. The development of the porcelain bacterial filter by Charles Chamberland (one of Pasteur’s collaborators and inventor of the autoclave), in 1884 made possible the discovery of viruses. Tobacco mosaic disease was the first to be studied with Chamberland’s filter. In 1892, a Russian Botanist, Dimitri Ivanowski published studies showing that leaf extracts from infected plants would induce tobacco mosaic disease even after filtration removed all bacteria. However, he attributed this to the presence of a toxin. 3 Martinus Beijerinck, a Dutch Microbiologist, working independently of Ivanowski, published the results of extensive studies on tobacco mosaic disease in 1898 and 1900. Because the filtered sap of diseased plants was still infectious, he proposed that the disease was caused by an entity different from bacteria, what he called a filterable virus. He observed that the virus would multiply only in living plant cells, but could survive for long periods in a dried state. Friedrich Loeffler and Paul Frosch in Germany found that the hoof-and- mouth disease of cattle was caused by a virus rather than by a toxin. In 1900, Walter Reed began his study of the yellow fever disease. Reed showed that this human disease was due to a virus (YFV) that was transmitted by mosquitoes in Cuba. Mosquito control soon reduced severity of yellow fever. Thus by the beginning of the 20th century, it had been established that viruses were different from bacteria and could cause diseases in plants, livestock, and humans. 4 In early 20th century, Vilhelm Ellermann and Oluf Bang in Copenhagen reported that Avian leukosis could be transmitted between chickens by cell-free filtrates and was probably caused by a virus (ALV). In 1911, Peyton Rous from the Rockefeller Institute in New York reported that a virus, now known as the Rous sarcoma virus (RSV), was responsible for a malignant muscle tumour in chickens. RSV is extensively used in cancer research in identification of oncogenes and targeted therapies. In 1915, Frederick Twort reported that bacteria could be attacked by viruses. Twort isolated bacterial viruses that could attack and destroy micrococci and intestinal bacilli. Although he speculated that his preparations might contain viruses, Twort did not follow up on these observations. It remained for Felix d’Herelle to establish decisively the existence of bacterial viruses. d’Herelle isolated bacterial viruses from patients with dysentery, probably caused by Shigella dysenteriae. 5 He noted that when a virus suspension was spread on a layer of bacteria growing on agar, clear circular areas containing viruses and lysed cells developed. A count of these clear zones allowed d’Herelle to estimate the number of viruses present. This procedure for enumerating viruses is now called a plaque assay. d’Herelle demonstrated that bacterial viruses could reproduce only in live bacteria; therefore he named them bacteriophages (or just phages) because they could eat holes in bacterial lawns. The chemical nature of viruses was established when Wendell Stanley announced in 1935 that he had crystallized the tobacco mosaic virus (TMV) and found it to be largely of protein. A short time later Frederick Bawden and Norman Pirie managed to separate the TMV virus particles into protein and nucleic acid. Thus by the late 1930s it was becoming clear that viruses are complexes of nucleic acids and proteins able to reproduce only in living cells. 6  General properties of viruses Viruses are a unique group of infectious agents. A complete virus particle or virion consists of one or more molecules of DNA or RNA enclosed in a coat of protein. Some viruses have additional layers that can be very complex and contain carbohydrates, lipids, and additional proteins. 7  Viruses can exist in two phases: extracellular and intracellular. Virions, the extracellular phase, possess rarely enzymes and cannot reproduce independent of living cells. In the intracellular phase, viruses exist primarily as replicating nucleic acids that induce host metabolism to synthesize virion components; eventually complete virions are released. Viruses differ from living cells in at least three ways; 1. Their simple, acellular organization. 2. The presence of either DNA or RNA, in almost all virions. 3. Their inability to reproduce independent of host cells (Obligate intracellular parasites). 8 Structure of viruses Virus morphology has been intensely studied using different techniques such as electron microscopy, X-ray diffraction, biochemical analysis, and immunology. Although our knowledge is incomplete due to the large number of different viruses, the general nature of virus structure is becoming clear. Virion size Virions size range from about 10 - 400 nm in diameter. The smallest viruses are a little larger than ribosomes (20 nm), whereas the poxviruses (vaccinia), are about the same size as the smallest bacteria (Mycoplasma genitalium - 0.3 μm). Most viruses are too small to be visible in the light microscope and must be viewed with scanning and transmission electron microscopes. 9 The size and morphology of selected viruses. The viruses are drawn to a scale of 1 μm10line. Structural properties of virion All virions consists of a nucleocapsid core (indeed, some viruses consist only of a nucleocapsid). The nucleocapsid is composed of a nucleic acid, usually either DNA or RNA, held within a protein coat called the capsid, which protects genetic material and aids in its transfer between host cells. Capsids are large macromolecular structures formed many copies of one or a few types of proteins. The proteins used to build the capsid are called protomers. Probably the most important advantage of this design strategy is that the information stored in viral genetic material is used with maximum efficiency. For eg., the tobacco mosaic virus (TMV) capsid is constructed using a single type of protomer that is 158 amino acids in length. Therefore, of the 6,000 nucleotides (nts) in the TMV genome, only about 474 nts are required to code for the coat protein. 11 The morphological types of viruses result from the combination of a particular type of capsid symmetry with the presence or absence of an envelope, which is a lipid layer external to the nucleocapsid. There are 3 types of capsid symmetry: helical, icosahedral, and complex. Those virions having an envelope are called enveloped viruses; whereas those lacking an envelope are called naked viruses. 12  a) b) Fig. Tobacco Mosaic Virus structure. a) An electron micrograph of the negatively stained helical capsid. b) Illustration of TMV structure. Note that the nucleocapsid is composed of a helical array of protomers with the RNA spiralling on the inside. 13 1. Helical capsids Helical capsids are shaped like hollow tubes with protein walls. Eg. TMV. In this virus, the helical or spiral arrangement of protomer produces a long, rigid tube, 15 - 18 nm in diameter by 300 nm long. The capsid encloses a spirally wound single stranded RNA (ss RNA) genome, which lies within a groove formed by the protein subunits. Not all helical capsids are as rigid as the TMV capsid. The influenza virus genome is enclosed in thin, flexible helical capsids that are folded within an envelope. The nucleic acid appears to determine helical capsid length because the capsid does not extend much beyond the end of the DNA or RNA. 14 2. Icosahedral capsids The icosahedron is a polyhedron with 20 equilateral triangular faces (sides) and 12 vertices (corners). The icosahedral capsid is the most efficient way to enclose a space. A few genes, or only one gene, can code for proteins that self-assemble to form the capsid. In this way a small number of genes can specify a large 3-dimensional structure. The capsids are constructed from ring or knob-shaped units called capsomers. Capsomers of 2 types: Pentamers (pentons) have 5 protomer subunits; hexamers (hexons) possess 6. Pentamers are usually at the vertices of the icosahedron, whereas hexamers generally form its edges and triangular faces. 15  Fig. Ten equilateral triangular faces in Adenovirus. Other 10 faces are on the opposite side (total 20 faces). 9 vertices are seen in figure. 3 on the other side. Total 12 vertices) The icosahedron is constructed of 42 capsomers. 16 The self-assembly of capsids is not fully understood but enzymatic activity is not required to link protomers together. However, non-capsid proteins provide a scaffolding upon which the protomers are assembled. In some RNA viruses, both the pentamers and hexamers of a capsid are constructed with same type of proteins which is composed of different proteins in other viruses. Although most icosahedral capsids appear to contain both pentamers and hexamers, in a small ds DNA virus, simian virus 40 (SV-40), capsid consists of only pentamers. The virus is constructed of 72 cylindrical pentamers with hollow centers. 5 flexible arms extend from the edge of each pentamer toward neighboring pentamers. The arms of adjacent pentamers twist around each other and act as ropes that tie the pentamers together. 17 SV 40 virus 18 3. Viruses with capsids of complex symmetry Some viruses possess complex symmetry. Eg. Poxviruses and large bacteriophages. The poxviruses are the largest of the animal viruses (400 × 240 × 200 nm in size) and can even be seen with a phase contrast microscope or in stained preparations. They possess a complex structure with an ovoid to brick-shaped exterior. Poxvirus nucleoid consists of a ds DNA associated with proteins in a central biconcave disk which is surrounded by a membrane. Two elliptical or lateral bodies lie between the nucleoid and its outer envelope. Outer envelop is a thick layer covered by an array of tubules or fibers. 19  T-even bacteriophages (T2, T4, & T6) that infect Escherichia coli have binal symmetry because they have a head that resembles an icosahedron and a tail that is helical. The icosahedral head contains the DNA genome. The tail is composed of a collar joining it to the head, a central hollow tube, a sheath surrounding the tube, and a complex baseplate. The sheath is made of 144 copies of the gp18 protein arranged in 24 rings, each containing 6 copies. In T-even phages, the baseplate is hexagonal and have tail pins and tail fibers joined at each corner of baseplate. T1, T5 odd phages, and lambda phages have sheathless tails that lack a baseplate and terminate in rudimentary tail fibers. T3 and T7 coliphages (phages that infect E. coli) have short, non-contractile tails without tail fibers. 20 Viral envelopes and enzymes Many animal viruses, some plant viruses, and bacterial viruses are bounded by an outer membranous layer called an envelope. Animal virus envelopes usually arise from host cell nuclear or plasma membranes; their lipids and carbohydrates are host constituents. In contrast, envelope proteins are coded by virus genes and may even project from the envelope surface as spikes, which are termed as peplomers. These spikes are involved in virus attachment to the host cell surface. Because they differ among viruses, they can be used to identify some viruses. The envelope is a flexible, membranous structure, so enveloped viruses are pleomorphic. However, the envelopes of bullet shaped rabies virus are firmly attached to the nucleocapsid and endow the virion with a constant, characteristic shape. 21 22 Generally, enveloped viruses are ‘ether sensitive’: i.e., Envelope is disrupted by solvents like ether, so that lipid-mediated activities are blocked or envelope proteins are denatured and rendered the virus inactive. Influenza virus envelop possess 2 types of spikes (10 nm in length); the spikes with enzyme neuraminidase, which functions in the release of mature virions from the host cell and the other spikes have hemagglutinin proteins, which can bind the virions to red blood cell (RBC) membranes and cause the RBC to agglutinate (clump together). This is called hemagglutination. Hemagglutinins participate in virion attachment to host cells. Spike proteins are generally glycoproteins. Matrix or M protein (non-glycosylated protein) is found on the inner surface of the envelope which stabilize virion. 23 A few virions possess enzymes. These enzymes are associated with the envelope or capsid (Eg. Influenza virus neuraminidase), but most viral enzymes are located within the capsid. The virus also carries an enzyme, RNA-dependent RNA polymerases that synthesizes RNA using an RNA template of the genome. Thus although viruses lack true metabolism and cannot reproduce independently of living cells, they may carry one or more enzymes essential for completion of life cycles. Viral Genomes Viruses employ all 4 possible nucleic acid types: single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA. All 4 types are found in animal viruses. Most plant viruses have ssRNA genomes, and most bacterial viruses contain ds DNA. The size of viral genetic material varies greatly. 24 25 The smallest genomes of MS2 and Q viruses are around 4,000 nts, just large enough to code for 3 or 4 proteins. These viruses save space by using overlapping genes. At the other extreme, (largest genome) T- even bacteriophages, herpes virus, and vaccinia virus have genomes of 1 - 2 × 105 nts and may direct the synthesis of over 100 proteins. DNA viruses: 1. Some DNA genomes can switch from one form to the other. For eg., λ phage of E. coli has a linear ds DNA genome in the capsid, but is converted into a circular form in the host cell. 2. Some DNA viruses genomes often contain unusual nitrogenous bases. For eg., T-even coli phages have 5-hydroxymethylcytosine (5’ HMC) instead of cytosine. 26 RNA viruses: ssRNA genome that is identical to viral mRNA in its base sequence is called the plus strand or positive strand. In fact, +ss RNAs can direct protein synthesis immediately after entering the cell. Eg. Polio, TMV, RSV. However, ssRNA genome that are complementary to viral mRNA is called the minus strand or negative strands. Eg. Rabies, mumps, measles, influenza viruses. -ssRNA virus synthesize RNA-dependent RNA polymerase to synthesize complimentary strand in replication. Many RNA viruses have segmented genomes, i.e., more than one RNA strand or segment (up to 10 - 12) and each segment codes for one protein and these segments are enclosed in the same capsid. Many plant and animal +ssRNA virus resembles eucaryotic mRNA as it has a 5′ cap of 7-methylguanosine (m7G) and a poly-A sequence at the 3′ end of their genome which helps in gene regulation and stability of mRNA. 27 28