Scientific Summary: A Study And Classification Of Viruses - Lecture Notes 1-12 PDF

Preview

Summary

Detailed summary on viruses, completed by Fiona Henrequz ...

Description

Viruses 1.

Introduction

Viruses are unlike all other living organisms in that they lack a cellular structure. They do not undergo growth and division but are constructed from component parts produced within organisms which they have infected. Viruses also lack the enzymes required to generate metabolic energy. Viruses are obligate intracellular parasites i.e. they require a suitable host cell to replicate within and are unable to do so on their own. Viruses are highly specific in the host cells which they require and there are viruses of animals, plants, fungi, protozoa and bacteria. Indeed, all other living organisms will have specific viruses which are capable of infecting them. (Chlamydia and Rickettsia grow and reproduce only within living host cells but are classed as bacteria since they have a cellular structure (membranes, ribosomes, metabolic pathways etc., contain DNA and RNA and reproduce by binary fission) One of the fundamental questions in virology is “are viruses alive?” This question is difficult to answer since they lack many of the features of all other living organisms and one view is that viruses are alive when they are located in a host cell (i.e. when they are infecting another organism) and inert when they exist outside of a host cell. Other virologists use the terms active and inactive to describe these two states. 2.

Classification of Viruses

Based on the 2000 report from the ICTV (International Committee on Taxonomy of Viruses) over 1,550 species of virus are now recognised. It is unknown how many other viruses are undiscovered at this point. Viruses are divided into 3 orders and within these orders specific families and species of virus fall within these groups. Groups I – VII are used to divide viruses on the basis of their genomes: Group I II III IV V VI VII

Genome dsDNA viruses ssDNA viruses dsRNA viruses (+) Sense RNA viruses (-) Sense RNA viruses RNA Reverse Transcribing viruses DNA Reverse Transcribing viruses

The subviral particles (satellites, viroids and prions) are placed in separate groups.

3.

Virus Structure

Extensive studies using electron microscopy, immunology and biochemical analysis have resulted in an increasingly clear picture of viral structure. The virus particle (or virion) is composed of: ▪ ▪ ▪

DNA or RNA (the genome) A protein coat (the capsid) In some cases an additional outer layer (the envelope)

Virions range in size from 10nm (not much larger than a ribosome) to 300-400 nm (about the size of the smallest bacteria). Some of the very largest viruses are visible using the light microscope but most require the use of the electron microscope. Viral Genome The vast majority of viruses contain a genome of DNA or RNA. The 4 possibilities are ssDNA (single stranded DNA), dsDNA (double stranded DNA), ssRNA (single stranded RNA) or dsRNA (double stranded RNA). Animal viruses may contain any one of these types of nucleic acid while plant viruses most often contain ssRNA and bacteriophages most often contain dsDNA. DNA viruses may possess linear or circular DNA. RNA viruses may contain RNA: ▪ ▪

Plus or Positive strand RNA – RNA which has an identical code to that of viral mRNA Minus or Negative strand RNA – RNA which has a complementary sequence to that of viral mRNA

RNA genes may be segmented whereby it is divided into separate parts which may even be present in separate virions. The smallest viral genomes are only large enough to code for 3-4 proteins while the largest genomes code for over 100. Capsid The nucleic acid and its surrounding protein coat are termed the nucleocapsid core. If a virus has no envelope it is termed naked while a surrounding envelope renders the virus enveloped. The capsid is composed of protein subunits called protomers. The protein capsid may have one of 3 general morphologies or symmetries. (i) (ii) (iii)

Helical Icosahedral Complex

Helical Capsids The protomers in a helical capsid are arranged in a helical or spiral arrangement. The genetic material is wound in a spiral in the middle of the capsid or in a groove inside the capsid. The capid’s overall morphology is a hollow tube with protein walls. An examples of a virus with a naked helical capsid is tobacco mosaic virus (figure 1) which causes symptoms including mottling, tissue death, stunting, yellowing and leaf curling in a range of plants (including tobacco and tomato plants).

Figure 1: Tobacco mosaic virus and infected plant (images from http://www.apsnet.org/education/LessonsPlantPath/TMV) There are no identified naked helical viruses which infect animals (or humans) although it is not clear why this should be the case other than it probably relates to host cell biology. Icosahedral Capsids Icosahedral capsids are regular polyhedrons with 20 equilateral triangle faces and 12 vertices (points). Icosahedral capsids are composed of protein units called capsomers with each composed of 5 or 6 protomers. Pentamers contain 5 protomers and hexamers contain 6 protomers. Pentamers are at the vertices of the polyhedron and hexamers form the edges and triangular faces. Icoasahedral capsids appear spherical when viewed at low magnification using an electron microscope. (Figure 2)

An example of a virus with a naked icosahedral capsid is the poliovirus (types 1 and 3) (figure 3). In most cases, poliovirus causes flu-like symptoms and is rapidly cleared up by the immune system. However, in about 1 in 100 cases, the virus spreads to the nerve cells that control muscle motion, causing paralysis and the condition known as poliomyelitis.

Figure 3: Polio virus (image from http://www.cat.cc.md.us/courses/bio141/lecguide/unit2/viruses/dkpolio.html)

Complex Capsids Some viruses possess capsids which are neither helical nor icosahedral. These include: ▪

Large bacteriophages with icosahedral heads, helical tails and possibly additional structures (fibres or “legs”) (figure 4). These viruses are said to show binal symmetry.

Figure 4 (image from http://www.agen.ufl.edu/~chyn/age2062/lect/lect_06/lect_06.htm)

•

Poxviruses (figure 5) with complex internal structures and ovoid to brick shaped exterior

Figure 5 Poxvirus (image from http://www.umanitoba.ca/faculties/medicine/medical_microbiology/Services_EM.htm)

Envelopes Many animal viruses, some plant viruses and several bacteriophages have an additional layer surrounding the capsid. This outer membrane is known as an envelope and normally consists of a lipid bilayer containing proteins and glycoproteins. Enveloped animal viruses normally obtain the envelope upon leaving the infected host cell. The lipids and carbohydrates in the envelope are obtained from the host cell’s nuclear or plasma membranes while the proteins are coded for by the viral genome. Envelope proteins may be external spikes (peplomers) as in the influenza virus (figure 6). The enveloped capsid may be helical (e.g. influenza) or icosahedral (e.g. HIV). Viral envelopes are flexible structures and consequently are of variable shape (pleomorphic). An example of an enveloped virus is the influenza virus (Figure 6). Influenza viruses possess envelope spikes which contain proteins (neuraminidase and haemagglutinin) involved in attachment and fusion with the host cell’s plasma membrane. In most forms of the influenza virus the genes coding for these surface proteins undergo mutations on a frequent basis (almost yearly with influenza A). This allows the influenza virus to cause epidemics or pandemics by evading the human immune system despite humans being continually exposed to influenza viruses.

Figure 6 Influenza virus (image from http://www.coloradoallergy.com/news/images/influenza_virus.gif)

4.

Sub-viral particles

It is now apparent that even simpler infectious particles than viruses exist. A brief summary of the major types follows. Viroids – small rod-like RNA molecules with no capsid or envelope. Several viroids are associated with plant diseases. Satellites – small RNA molecules which act as parasites of viruses. Satellites require the presence of the “helper” virus to replicate within a host and cause additional symptoms beyond those observed if the virus alone is present. These agents are primarily associated with plant viruses (e.g. the barley yellow dwarf virus satellite). Hepatitis delta virus (HDV) is a unique RNA agent which causes disease in humans. It possesses some viroid and some satellite properties. HDV requires the presence of Hepatitis B virus (HBV) as a “helper” virus to replicate. HDV appears to increase the mortality rate associated with HBV. Prions (proteinaceous infectious particles) Prions would appear to consist only of protein with no genome. The manner in which prions cause disease is not entirely clear although one proposed mechanism involves the infectious prion replacing normal proteins present in the host’s brain. This results in the formation of plaques, tissue damage and a breakdown in neurological function. Examples of diseases caused by prions include scrapie, bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD) and Kuru.

4.

Viral Replication

There are a number of variations on the process of viral infection and replication within host cells. This section will provide details of a number of specific viral infection cycles. Bacteriophages have been studied extensively by virologists to provide a model of viral replication. Work on bacteriophage replication was conducted as far back as 1939 and highlighted the 3 major stages of viral replication: ▪ ▪ ▪

Initiation of infection Replication and expression of the virus genome Release of mature virions from the infected cell

The process of replication is often divided into a number of stages for ease of explanation. It should be noted that these stages are more identifiable as distinct stages in some viruses than they are in others and certain stages may appear to occur simultaneously in some instances. The stages are: ▪ ▪ ▪ ▪ ▪ ▪ ▪

Attachment Penetration Uncoating Genome replication and gene expression Assembly Maturation Release

Attachment Virus attachment involves the specific binding of a virus-attachment protein (also termed antireceptor) to a cellular receptor molecule on the host cell. Animal viruses bind to plasma membrane proteins (often glycoproteins) or carbohydrate residues on glycoproteins/glycolipids. Protein receptors tend to be more specific than carbohydrate group receptors. Some complex viruses (poxviruses, herpesviruses) utilise more than one receptor and therefore have several routes of uptake into host cells. It is important to realise that cellular receptor molecules have not evolved (and are not produced) by cells to allow viruses to enter. Rather, it is viruses which have taken advantage of molecules required by cells for normal cellular function. In some instances interactions with more than one protein are required for virus entry (i.e. these are not alternative receptors since both are required to effect virus entry). The distribution of receptor proteins in different tissues and organisms plays a major role in determining which hosts and tissues specific viruses are capable of infecting (i.e. the tropism of the virus). Bacteriophages attach to bacterial host cells at specific receptor sites including LPS, cell wall proteins, teichoic acids, flagella and pili dependent on the specific virus. Plant viruses face greater difficulties due to the presence of the thick cellulose wall and protective layer of

pectin and waxes. Plant viruses are believed to rely on damage to the cell wall in order to gain entry rather than binding to receptor molecules. Penetration Penetration usually occurs rapidly after attachment. This stage generally requires energy and therefore the cell must be metabolically active. Four major mechanisms of penetration are currently recognised: ▪

Translocation of the entire virion across the cytoplasmic membrane. This mechanism is rare and not well understood. The process appears to be mediated by proteins in the capsid and specific membrane receptors.

▪

Most enveloped viruses enter cells via endocytosis. The virions attach to clathrin (a protein) coated pits on the cell membrane. This mechanism does not require any specific proteins and relies on a normal formation and internalisation of coated pits at the cell membrane. These pits are employed by the cell to internalise a wide range of materials (including proteins, growth factors and toxins). The pits pinch off from the membrane and form coated vesicles in which the virus is engulfed. Within seconds most of these vesicles will fuse with endosomes and release their contents into these larger vesicles (at this point the virus has still not strictly entered the cell (or at least its cytoplasm)). Endosomes fuse with lysosomes (organelles involved in intracellular digestion of substances). Due to the presence of degradative enzymes and an acidified interior the virus must enter the cytoplasm before being degraded. Exit from the lysosome may involve membrane fusion (see below for mechanism).

▪

Some enveloped viruses (e.g. influenza) fuse with the cell membrane. This process requires the presence of specific fusion proteins in the viral envelope which promote joining of cellular and viral membranes. The nucleocapsid is deposited into the cytoplasm of the host cell.

▪

Some bacteriophages (e.g. T4 phage) inject their DNA into the cell through a tube which penetrates the bacterial cell wall. Some non-enveloped animal viruses (e.g. the poliovirus) undergo structural changes following adsorption and only the nucleic acid enters the cell.

Uncoating Uncoating refers to the events following penetration when the capsid is completely or partially removed to expose the genome. Uncoating is not as fully understood as some of the other stages of viral replication. The product of uncoating depends upon the specific virus. ▪ ▪ ▪

Picornaviruses uncoat to produce a 23 amino acid protein attached to the RNA genome. Retroviruses have complex cores with an RNA genome and the enzyme reverse transcriptase. Poxviruses do not completely uncoat.

Removal of a viral envelope during membrane fusion is part of the uncoating process

Genome replication and gene expression The replication process depends upon the type of genome possessed by a specific virus. These different genomes are also the basis of classifying viruses into 7 groups as proposed by David Baltimore in 1971. (see earlier) The T4 phage of E. coli has been extensively studied with regards to its replication cycle. Shortly after phage DNA has been injected the host cell ceases to synthesise DNA, RNA and protein and is forced to produce viral components. E. coli RNA polymerase starts to produce viral mRNA instead of bacterial mRNA. This early mRNA (the mRNA produced before phage DNA is produced) directs the synthesis of proteins and enzymes which allow the virus to take over the host cell and force it to produce viral nucleic acids. Some early genes code for enzymes which break down E. coli DNA. This degradation of host DNA ceases the expression of host genes and provides nucleotides for the synthesis of phage DNA. (Animal viruses do not often degrade the host DNA. Early mRNA is produced by a viral polymerase (and not the host’s enzymes) in the case of poxviruses.) Late mRNA (produced after the replication of phage DNA) direct the synthesis of structural proteins, proteins involved in phage assembly and proteins involved in phage release from the host cell. Viruses which have an RNA genome show even more variations in their replication cycle. In these viruses: ▪ ▪ ▪

The RNA genome may act as the mRNA to be directly translated as a protein. These genomes are (+) sense RNA. The RNA genome may act as a template for mRNA synthesis by a viral enzyme. These are (-) sense RNA genomes. The RNA may be converted to a DNA intermediate by reverse transcriptase. This DNA can direct the synthesis of mRNA. This system is seen in retroviruses such as HIV.

Assembly Assembly involves the collection of all of the viral components to produce a complete virion. In T4 phage many of these steps occur spontaneously although some proteins may be required. This is also true for animal viruses where capsid self-assembly has been described. The genome can be surrounded by a capsid as it forms or inserted into the capsid core after it has assembled. The process of nucleic acid insertion is still unclear.

Maturation Stage at which the virus becomes infectious and may involve structural changes in the capsid proteins. Release The mechanism of release differs between non-enveloped and enveloped viruses. Naked viruses are often released by cell lysis and hence infection results in cell destruction. Enveloped viruses are released by budding whereby the nucleocapsid passes through the cell membrane. The envelope consisting of membrane lipids and viral proteins is obtained during this process.

“LIFECYCLES”. 1.

Examples of specific viral “Life cycle”

(a) Poliovirus

(Image from http://webs.wichita.edu/mschneegurt/biol103/lecture12/lecture12.html)

The polio virus binds to CD155 (an immunoglobulin-like molecule). This molecule sticks out from the surface of the cell and lies across the surface of the poliovirus capsid at a “canyon”. The virus enters the host cell by endocytosis. Translation of the ssRNA genome (which acts as mRNA) into a single polyprotein occurs with subsequent cleavage (by virus coded proteases) into separate proteins (including RNA proteases responsible for shutting off the host cell’s protein synthesis thereby freeing more ribosomes to translate the viral genome and ensuring the host cell will ultimately die to release virus particles). Replication is catalysed by a RNAdirected RNA polymerase released from the polyprotein during cleavage. The initial round of replication produces a single complementary RNA strand which is then used to produce a +ve sense copy of the original genome. Capsid proteins self-assemble and, by a mechanism which is not fully understood, the viral RNA enters the capsid. The mature virions are released by cell lysis. (b) HIV

(image from http://www.thebody.com/nmai/cycle.html)

Once inside the body a viral envelope protein interacts with a glycoprotein plasma membrane receptor present on CD4+ T cells, macrophages, dendritic cells and monocytes. Fusion of the envelope with the plasma membrane occurs and the viral core (and two RNA strands) enter the cell cytoplasm. Viral reverse transcriptase catalyses the synthesis of a DNA strand from the viral RNA. Ribonuclease H degrades the RNA and the DNA strand is duplicated to produce a double stranded DNA copy of the original RNA genome. The dsDNA is integrated into the host genome (this step is catalysed by the HIV integrase enzyme) as a provirus. Provirus can force the cell to synthesise viral mRNA. Host ribosomes translate viral mRNA to produce viral proteins. Proteins and viral RNA genome are assembled in the cytoplasm and the virus exits the host cell by budding through the plasma membrane.

(c) HSV (Herpes Simplex Virus)

Herpes viruses are a large group of enveloped viruses with icosahedral capsids and dsDNA genomes. The virus attaches to a glycoprotein receptor on the cell surface. Virus enters cell following fusion of envelope with cell membrane. The nucleocapsid is transported to the nuclear pores and the viral DNA is released into the nucleus. Viral genome replicates in the nucleus. A viral “host shut off protein” remains in the cytoplasm. Protein synthesis occurs in the cytoplasm on the host cell ribosome. After entering the host cell the genome circularises, immediate early genes are transcribed followed by early and late. Pr...