Virology. Virology: virus replications, ssrna viruses, Plant viruses, Sanitation

Transcription

1 Virology Virology: virus replications, ssrna viruses, Plant viruses, Sanitation

2 VIRUSES-definition A virus is a non-cellular, obligate intracellular parasite which has two phases in its life cycle: 1. An extracellular phase in which it exists as a virion, Virion :a particle consisting of the genomic nucleic acid (DNA or RNA) molecule or molecules surrounded by a protective shell of protein (sometimes with lipid) called the capsid or coat. 2. An intracellular phase in which the viral genome (no longer encapsidated) is in intimate contact with the components of a living host cell. During this phase, viral replication can occur. Viral replication involves the assembly of pre-formed viral components, not growth nor binary fission.

3 Viruses are energy parasites Virus particles (virions) themselves do not grow or undergo division Viruses lack the genetic information which encodes apparatus necessary for the generation of metabolic energy or for protein synthesis (ribosomes) No known virus has the biochemical or genetic potential to generate the energy necessary to drive all biological processes (e.g. macromolecular synthesis). They are absolutely dependent on the host cell for this function.

4 Virus diversity There is more biological diversity within viruses than in all the rest of the bacterial, plant & animal kingdoms put together. This results from the success of viruses in parasitizing all known groups of living organisms. Viruses are not usually classified into conventional taxonomic groups but they are usually grouped according to such properties as: size the type of nucleic acid they contain the structure of the capsid & the number of protein subunits in it host species immunological characteristics

5 Virus particle Nucleic acid surrounded by a protein coat Nucleic acid is infective portion Protein coat protects nucleic acid Only enough nucleic acid for a few genes Depends on host cell for all other constituents for replication Particle Shapes Rods Spherical (icosahedral) Bacilliform (bullet-shaped)

6 Flexuous rods Spherical (icosahedral) Rigid rods Bacilliform shape viruses

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8 The Baltimore Classification : Genome Replication & Gene Expression The replication strategy of any virus depends on the nature of its genetic material. All viruses can be divided into seven groups - such a scheme was first proposed by David Baltimore in Originally, this classification included only six groups, but it has since been extended to include the scheme of genome replication used by the hepadnaviruses & caulimoviruses. The central theme here is that all viruses must generate positive strand mrnas from their genomes, in order to produce proteins and replicate themselves. David Baltimore, who originated the scheme, has given his name to the so-called "Baltimore Classification" of virus genomes.

9 The Baltimore Classification Based on replication strategy, viruses can be classified into seven (arbitrary) groups: 1. Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses) 2. Single-stranded (+)sense DNA (Parvoviruses) 3. Double-stranded RNA (Reoviruses; Birnaviruses) 4. Single-stranded (+)sense RNA (Picornaviruses; Togaviruses, etc) 5. Single-stranded (-)sense RNA (Orthomyxoviruses, Rhabdoviruses) 6. Single-stranded (+)sense RNA with DNA intermediate (Retroviruses) 7. Double-stranded DNA with RNA intermediate (Hepadnaviruses)

10 The Virus Replication Cycle General Concepts

11 Virus Replication For a virus to multiply it must obviously infect a cell. Viruses usually have a restricted host range (i.e. animal and cell type in which this is possible. All must make proteins with 3 sets of functions: ensure replication of the genome package the genome into virus particles alter the metabolism of the infected cell so that viruses are produced.

12 Virus Replication Steps in the Replicative Cycle of viruses are still composed of these steps: 1. Attachment/Adsorption 2. Penetration 3. Uncoating 4. Biosynthesis (Genome replication & Synthesis of viral proteins) 5. Assembly 6. Release 7. Maturation

13 The Replication Cycle-Introduction Virus replication can be divided into stages, that are purely arbitrary divisions, used for convenience in explaining the replication cycle of a non-existent 'typical' virus. Regardless of their hosts, all viruses must undergo each of these stages in some form to successfully complete their replication cycles. Not all the steps described here are detectable as distinct stages for all viruses; often they blur together & appear to occur almost simultaneously.

14 The Replication Cycle Attachment; Penetration ; Uncoating ; Biosynthesis; Assembly ; Release ; Maturation

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18 Attachment/Adsorption Virus attachment consists of specific binding of a Viral Attachment Protein (VAP) to a Cellular Receptor. Many examples of virus receptors are now known. Receptor molecules may be: Proteins (usually glycoproteins - specific molecules), or Sugar residues present on glycoproteins or glycolipids (less specific). Some complex viruses (e.g. Poxviruses, Herpesviruses) may have more than one receptor/receptor-binding protein, therefore, there may be alternative routes of uptake into cells.

19 Attachment/Adsorption A viral receptor is defined as the cellular molecule to which a virus attaches to initiate replication. Attachment is in most cases a reversible process. This mean if penetration does not ensue, the virus can elute from the cell surface. Some viruses have specific mechanisms for "detachment, but elution from cell often leads to changes in the virus which decrease or eliminate the possibility of attaching to other cells.

20 Finding The Right Host Cell should be both in order to ensure the infection: Susceptible: it depends on the cell and the virus surface receptor interactions. Permissive: it refers for the viral replication within the cell and that depends on the intracellular components found only in certain cell types TROPISM of most viruses is referred to the expression (or absence) of receptors on the surface of cells, that determine the type of cell in which the viruses are able to replicate. It is important factor in pathogenesis. Note: viruses have no means for locomotion. Their propagation is dependent on the random encounter with potential host cells.

21 Virus Receptors Many examples of virus receptors are now known. Virus receptors fall into may different classes, e.g: immunoglobulin-like superfamily molecules (IgG-like) membrane-associated receptors transmembrane transporters & channels (Integrins) The one factor which unifies all virus receptors is that they did not evolve & are not manufactured by cells to allow viruses to enter cells.

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23 Multiple Receptors: Receptors and co-receptors In some cases, interactions with more than one protein (coreceptors) are required for virus entry. Neither protein alone is a functional receptor - both are required to act in concert. The primary receptor for HIV is the helper T cell differentiation antigen, CD4. It was found that there must be one or more accessory factors in addition to CD4, to form a functional HIV receptor. These are a family of proteins known as Chemokine receptors.

24 PENETRATION The virus entry into cells depends largely upon the TYPE of the cell, and of the virus: the cell type has a great deal of influence on the strategy the virus uses to gain access; in turn, specific virus types may employ different strategies to gain access to the same cell type. However, the greatest commonality in strategy is probably observed between viruses infecting a single broad type of host, defined by the nature of their cell walls. The precise biophysical details of the fusion process remain unknown

25 Viral Entry into Animal Cells Unlike attachment, viral penetration is an energy-dependent process, i.e. the cell must be metabolically active for this to occur. Three mechanisms may be involved: 1. TRANSLOCATION of the entire virion across the cell membrane, 2. ENDOCYTOSIS of the virus into intracellular vacuoles; eventually into the cytoplasm. 3. FUSION of the viral envelope with the cell membrane. Requires the presence of a viral fusion protein in the virus envelope, e.g. influenza haemagglutinin; retrovirus envelope glycoprotein. These mechanisms are shown in three different infection cycles for RNA or DNA viruses.

26 1-Translocation Translocation of the entire virus particle across the cytoplasmic membrane of the cell. This process is relatively rare among viruses & is poorly understood. It is mediated by proteins in the virus capsid & specific membrane receptors.

27 2-Endocytosis Endocytosis of the virus into intracellular vacuoles is probably the most common mechanism of virus entry into cells. Fusion does not require any specific virus proteins (other than those already utilized for receptor binding) but relies on the normal formation & internalization of coated pits at the cell membrane. Receptor-mediated endocytosis is an efficient process for taking up & concentrating extracellular macromolecules.

28 3-Fusion Fusion of the virus envelope (enveloped viruses only) with the cell membrane, either directly at the cell surface or in a cytoplasmic vesicle. Fusion requires the presence of a specific fusion protein in the virus envelope, e.g: Influenza haemagglutinin Retrovirus transmembrane glycoproteins, which promotes joining of the cellular & virus membranes There are two types of virusdriven membrane fusion: ph-dependent & ph-independent.

29 Graphic of Viral Entry into Animal Cells Virus attachment to cell surface (adsorption) and virus entry into cell. Picture shows translocation, pore formation, receptor mediated endocytosis using clathrin coated vesicles and membrane fusion. Some viruses can use more than one strategy

30 Plant viruses

31 Tobacco Mosaic Virus researchers independently described an unusual agent that caused mosaic disease in tobacco. This agent, later named Tobacco mosaic virus (TMV), was the first virus to be described. Since then, a large number of diverse viruses have been found in plants, animals, fungi, and bacteria

32 Adolf Mayer - the concept of transmissibility and the earliest concept of an ultra-filterable virus Adolf Eduard Mayer ( ) Tobacco mosaic disease, Mayer s work, 1866 Agents that pass through filters that retain bacteria came to be called ultra-filterable viruses, appropriating the term virus from the Latin for "poison".

33 1935-Wendell Stanley - purification and crystallization of tobacco mosaic virus Wendell Meredith Stanley ( ) Top: TMV, purified, negative contrast electron microscopy, minimum exposure study, Robley

34 PLANT VIRUSES Approximately 25-30% are plant pathogens, remainder animal pathogens or pathogens of other organisms The current estimate of recognized viruses is approaching 4,000, of which about 1,000 are plant viruses. The main reason that we study plant viruses is the negative impact that viral diseases have on crop production

35 Virus Infections of Plants Clearly, there are major differences between virus infections of plants & those of vertebrates. In economic terms, viruses are only of importance if it is likely that they will spread to crops during their commercial lifetime, which of course varies greatly between very short extremes in horticultural production & very long extremes in forestry. Some estimates put total worldwide damage due to plant viruses as high as US$ 6x1010 per year. The mechanism by which plant viruses are transmitted between hosts is of great importance.

36 Plant-Virus Interactions Recent progress in understanding virus-host interactions has transformed viruses into important tools of biomedicine and biotechnology. Plant viruses are being used to produce large quantities of proteins of interest in plants and to develop safe and inexpensive vaccines against human and animal viruses. Host ranges of individual viruses vary from very narrow to very broad e.g. :Cucumber mosaic virus infects over 1000 species in 85 plant families.

37 Resistance of plant species to viruses Resistance of plant species to viruses is determined primarily by the plant genotype. Plants possess active and passive means of preventing virus infection Passive defenses are due to the failure of the plant to produce one or more host factors required for virus reproduction and spread within the host. Active defenses include detection and destruction of the virus-infected cells due to the function of specific resistance genes in the plant. Resistance genes are active only against a particular virus.

38 Plant Virus Infections Typically, virus infections of plants might result in effects such as growth retardation, distortion, mosaic patterning on the leaves, yellowing, wilting, etc. These macroscopic symptoms result from: Necrosis of cells, caused by direct damage due to virus replication Hypoplasia, i.e. localized retarded growth frequently leading to mosaicism (the appearance of thinner, yellow areas on the leaves) Hyperplasia, which is excessive cell division or the growth of abnormally large cells, resulting in the production of swollen or distorted areas of the plant.

39 Viral Symptoms Examples small necrotic or chlorotic spots called local lesions. Pinto bean leaves with necrotic local lesions caused by Tobacco mosaic virus

40 In most cases, viruses spread throughout the whole plant and cause a systemic infection. Typical leaf symptoms of viral diseases include: mosaic patterns

41 yellowing, stripes or streaks

42 vein clearing, vein banding leaf rolling and curling

43 changes in the color of the flowers including : dramatic color mosaics called color breaking

44 Fruit and vegetable symptoms may include mosaic patterns, discoloration and chlorotic ringspots

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46 Stems of plants may develop tumors in response to virus infection

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52 Plant Virus Infections Initially, most plant viruses multiply at the site of infection, giving rise to localized symptoms such as necrotic spots on the leaves. The virus may subsequently, be distributed to all parts of the plant either by direct cell-to-cell spread or by the vascular system, resulting in a systemic infection involving the whole plant. However, the problem these viruses face in re-infection & recruitment of new cells is the same as they face initially - how to cross the barrier of the plant cell wall. Plant cell walls necessarily contain channels called plasmodesmata which allow plant cells to communicate with each other & to pass metabolites between them.

53 Movement Proteins Plasmodesmata channels are too small to allow the passage of virus particles or genomic nucleic acids. Many (if not most) plant viruses have evolved specialized movement proteins which modify the plasmodesmata: One of the best known examples of this is the 30k protein of tobacco mosaic virus (TMV). This protein is expressed from a subgenomic mrna & its function is to modify plasmodesmata causing genomic RNA coated with 30k protein to be transported from the infected cell to neighbouring cells. Other viruses, such as cowpea mosaic virus (CPMV, Comoviridae) have a similar strategy but employ a different molecular mechanism. In CPMV, the 58/48k proteins form tubular structures allowing the passage of intact virus particles to pass from one cell to another.

54 Movement Proteins

55 Replication Most of plant virus genomes are composed of ssrna that is the same (positive-sense) polarity as the messenger RNAs of the cell. However, the majority of plant viruses do not use DNA at all. Some of the plant viruses have genomes that are composed of single-stranded (ss) DNA. minority of plant viruses possess dsdna genomes

56 Replication Replication cycles start by penetration of the virion into the cell. Plant viruses are unable to penetrate the plant cuticle and cell wall. It is believed that the virion enters the cytoplasm of the cell passively through wounds caused by mechanical damage to the cuticle and cell wall. The next phase of virus infection is the partial or complete removal of the coat protein shell of the virion in the cytoplasm.

57 Next the cell mediates expression of the viral genome by providing a transcription apparatus (for DNA viruses) and a translation apparatus (for all viruses). The DNA viruses must be transported to the nucleus for transcription in order to gain access to the cell proteins required for the production of messenger RNA from viral DNA.

58 Translation of viral RNA in the cytoplasm produces viral proteins that are required for completion of the virus life cycle. The next step in the virus reproduction cycle is movement of the virus into neighboring cells through small channels called plasmodesmata that form connections between plant cells.

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60 VIRUS REPLICATION The majority (but not all) of plant virus families have (+)sense RNA genomes. +ssrna virus acts like mrna in host cell ssrna (+) Viruses virions ssrna(+) 1 5 (-)RNA early protein replicative 2 protein structural complex 4 3 mrna

61 (+)Sense RNA Plant Viruses The genome of Tobacco mosaic virus (TMV) is a wellstudied example - a 6.4 kb RNA molecule which encodes four genes. There is a 5' methylated cap & 3' end of the genome contains extensive secondary structure, but no poly(a) sequences. Expression is by producing non-structural proteins by direct translation of the open reading frame encoded in the 5' part of the genome & the virus coat protein & further non-structural proteins from two subgenomic RNAs encoded by the 3' part.

62 The TMV Genome

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64 Transmission of Plant Viruses Many bacterial & animal viruses possess the ability to facilitate their own entry into a host cell: PLANT VIRUSES DO NOT. Plant viruses must come in contact with host cytoplasm (can t grow or move) Therefore, plant viruses must enter their hosts through : Wounds (Mechanically Transmitted) or be Vectored by other organisms.

65 Transmission of Plant Viruses Mechanical Transmission. Passage through Plant Material - Through seeds, grafting and vegetative propagation. - Parasitic plants. Transmission through Fungi /Protists. Transmission through Invertebrates. - Nematodes - Insects: Aphids, Sucking Insects, & Beetles Artificial Modes of Transmission.

66 Mechanical Inoculation (Abrasion) Most Widely Used Experimental Method Can occur in agriculture: contact of plants with infected plants, or plant material, contact with contaminated implements, or poor hygiene. On leaves... estimates for the minimum number of virions per infection event (lesion) can vary greatly: e.g TYMV virions/lesion; 450 TMV virions/lesion.

67 Transmission Through Plant Material Soil debris (e.g. TMV) Seeds & Pollen: About 20% of plant viruses - Present on seed coats and infect seedlings during germination (e.g.tmv on tomato seeds). Vegetative propagation (e.g. potato). Grafting (esp. in transmission of fruit tree viral diseases). Dodder ( Cuscuta spp.) is a parasitic vine. It can transmit a wide range of viruses between many different species of host plants. [Used to be a widely used Dodder

68 Transmission By Fungi /Protists Zoosporic fungi e.g. Olpidium spp. transmit isometric viruses like TNV (along with STNV), cucumber necrosis virus.

69 Transmission Through Invertebrates: - Nematode No known plant viruses replicate in the nematode vectors. The virus particles attach to the stylet and lining of the buccal cavity. The retention of viruses (which can be up to a year) in nematode vectors is controlled by the viral Coat Protein. Examples of nematode-transmitted viruses.. Xiphenema index transmit GFLV

70 Transmission Through Invertebrates: - Insects Viruses are transmitted predominantly by sucking insects like aphids, but some are transmitted by chewing insects like beetles, or even pollinators like bees. The most important group of viral vectors among the insects are the Homoptera: -Aphids: The best studied are: Myzus persicae and Aphis gosypiae -Whiteflies -Leafhoppers -Mealybugs Aphids and related insects are the single most important group of vectors. About 66% of the approx 400 viruses known to have

71 Transmission Through Insects *Longtailed mealybug *Taillike filaments *Obscure mealybug *Grape mealybug. Bemisia tabaci

72 Insect as a Vector Insects which bite or suck plant tissues are the ideal means of transmitting viruses to new hosts - "nonpropagative transmission". However, in other cases (e.g. many plant Rhabdoviruses) the virus may also infect & multiply in the tissues of the insect ("propagative transmission") as well as those of host plants. In these cases, the vector serves as a means not only of distributing the virus, but also of amplifying the infection.

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75 Helper Components (or Helper Factors) For most aphid transmitted viruses, CP is the most important determinant of transmission. But...In some cases other viral genes may encode essential transmission factors. (e.g. the ORF II gene protein of CaMV). Sometimes, the transmission factor of one virus can aid the transmission of another by aphids (e.g. members of the potyvirus family)--- in these cases the transmission factor is termed a "Helper Component or Helper Factor, (e.g. HC-Pro)

76 Helper Components (or Helper Factors) What do the helper component-proteinase do? The Bridge hypothesis. binding to virions and to aphid mouthparts thus allowing retention of virions at a site from which they can subsequently be inoculated (Govier & Kassanis, 1974). Several studies support the bridge hypothesis (Berger & Pirone, 1986;Ammaret al., 1994; Wanget al., 1996; Blancet al., 1997), but there is little information about the location of the virion-binding or stylet-binding functions on the HC molecule The helper component-proteinase (HcPro) mediates the attachment of a virus to the vector. Specific amino acid domains in both potyvirus CP and HcPro are required for aphid transmission: CP:HcPro binding in vitro Virus transmission

77 Artificial/experimental Means Of Inoculation 1. Protoplasts: Useful for experiments to investigate virus replication (synchronous infection) or to distinguish between resistance mechanisms that affect movement versus replication. Methods: Isolation of protoplasts from plant tissue or tissue culture using macerase (pectinase) and cellulase. Inoculation of protoplasts with viral nucleic acid by electroporation or with nucleic acid or virions using polyethylene glycol.

78 Artificial/experimental Means Of Inoculation 2. Agroinfection. Useful for infection of plants with viruses that are difficult to transmit efficiently, e.g. certain geminiviruses, or when you need to deliver a very strong pulse of viral nucleic acid, e.g. in virus-induced gene silencing Methods: 1. Clone virus sequence into a T-DNA vector under the control of a strong constitutive promoter. 2. Transform Agrobacterium tumefaciens with the construct. 3. Infiltrate a suspension of the A. tumefaciens into leaf tissue (gene silencing) or needle-inoculate meristem area (geminivirus work). 4. Alternatively, use the gene gun to fire in the T-DNA vector.

79 Feeding behavior Feeding behavior also influences virus transmission: The vector selects or tests hosts by brief exploratory probes into multiple epidermal cells. More prolonged feeding takes place if the host is suitable and the aphid searches for the carbohydrate rich phloem sap. The initial shallow probes mean that vector and virus need not share the same host. This kind of feeding is sufficient for inoculation of non-circulatively, non-persistantly vectored viruses. Deep phloem feeding allows transmission of circulatively as well as non-circulatively transmitted viruses.

80 Diagnosis of Plant Virus Diseases 1. Pathogenicity Bioassays using indicator plants. 2. Transmissibility Vector transmission assays. Because of vector specificity, identification of the organism that transmits the virus may provide important information for virus identification. 3. Architecture of virus particles Electron microscopy. The shape and size of virions distinguish rod-shaped, filamentous, icosahedral, or large enveloped particles.on the other hand, viruses sharing the same shape and size are difficult to distinguish by their appearance. For instance, small spherical viruses may be difficult to distinguish from each other and from plant ribosomes. 4. Properties of the protein coat Immunological procedures, scientists have taken advantage of this specific interaction to develop laboratory tests for plant viruses. 5. Properties of viral nucleic acid PCR amplification,

81 Sanitation Various approaches have been applied to eliminate viruses in Plants: Chemotherapy Cryotherapy Meristem-tip culture Micrografting Somatic embryogenesis

82 Example of Plant Viruses Top 10 plant viruses in molecular plant pathology Top 10 based on scientific/ economic importance: 1. Tobacco mosaic virus (TMV) Karen-Beth G. Scholthof 2. Tomato spotted wilt virus (TSWV) Scott Adkins 3. Tomato yellow leaf curl virus (TYLCV) Henryk Czosnek 4. Cucumber mosaic virus (CMV) Peter Palukaitis 5. Potato virus Y (PVY) Emmanuel Jacquot 6. Cauliflower mosaic virus (CaMV) Thomas Hohn and Barbara Hohn 7. African cassava mosaic virus (ACMV) Keith Saunders 8. Plum pox virus (PPV) Thierry Candresse 9. Brome mosaic virus (BMV) Paul Ahlquist 10. Potato virus X (PVX) Cynthia Hemenway

83 Viruses just missing out on the Top 10, including 1. Citrus tristeza virus (CTV) 2. Barley yellow dwarf virus (BYDV) 3. Potato leafroll virus (PLRV) 4. Tomato bushy stunt virus (TBSV)