What is a Virus?

 

 

Viruses

Viruses may be defined as acellular organisms whose genomes consist of nucleic acid, and which obligately replicate inside host cells using host metabolic machinery and ribosomes to form a pool of components which assemble into particles called VIRIONS, which serve to protect the genome and to transfer it to other cells.

They are distinct from other so-called VIRUS-LIKE AGENTS such as VIROIDS and PLASMIDS and PRIONS

 

Alternative definitions:

 Viruses 1:

"Viruses are entities whose genomes are elements of nucleic acid that replicate inside living cells using the cellular synthetic machinery, and cause the synthesis of specialised elements [virions] that can transfer the genome to other cells".

SE Luria, JE Darnell, D Baltimore and A Campbell (1978). General Virology, 3rd Edn. John Wiley & Sons, New York, p2 of 578.

 

Viruses 2:
"Virus are submicroscopic, obligate intracellular parasites...[and]

·         Virus particles (virions) are formed from the assembly of pre-formed components;

·         Virus particles 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 (eg: ribosomes)".

AJ Cann (1997). Principles of molecular virology, 2nd Edition. Academic Press, San Diego.

 

The concept of a virus as an organism challenges the way we define life:

• viruses do not respire,
• nor do they display irritability;
• they do not move
• and nor do they grow,
• however, they do most certainly reproduce, and may adapt to new hosts.

By older, more zoologically and botanically biased criteria, then, viruses are not living. However, this sort of argument results from a "top down" sort of definition, which has been modified over years to take account of smaller and smaller things (with fewer and fewer legs, or leaves), until it has met the ultimate "molechisms" or "organules" - that is to say, viruses - and has proved inadequate.

Classical Properties of Living Organisms:

·         Reproduction

·         Nutrition

·         Respiration

·         Irritability

·         Movement

·         Growth

·         Excretion

More modern definitions include the storage and replication of genetic information as nucleic acid, and the presence of or potential for, enzyme catalysis

(Penguin Dictionary of Biology, 9th Edition, 1994)

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Newer Definitions of Life

Life may (somewhat irreverently) be defined in general terms as:

"The phenomenon associated with the replication of self-coding informational systems",

 

or more specifically as:

"The phenomenon associated with the replication of nucleic acids".

- Rybicki EP, 1996.

 

Another more serious view:

"Life can be viewed as a complex set of processes resulting from the actuation of the instructions encoded in nucleic acids. In the nucleic acid of living cells these are actuated all the time; in contrast, in a virus they are actuated only when the viral nucleic acid, upon entering a host cell, causes the synthesis of virus-specific proteins. Viruses are thus "alive when they replicate in cells, while outside cells viral particles are metabolically inert and are no more alive than fragments of DNA."

- Dulbecco R and Ginsberg HS, 1980. Virology, p.854-855 (originally published as a section in Microbiology, 3rd Edn., Davis et al., Harper and Row, Hagerstown).

 

Copyright Ed Rybicki, October 1995; April, June,1998

If one defines life from the bottom up - that is, from the simplest forms capable of displaying the most essential attributes of a living thing - one very quickly realises that the only real criterion for life is:

 

The ability to replicate

and that only systems that contain nucleic acids - in the natural world, at least - are capable of this phenomenon. This sort of reasoning has led to a new definition of organisms:

Organism:
"An organism is the unit element of a continuous lineage with an individual evolutionary history."

SE Luria, JE Darnell, D Baltimore and A Campbell (1978). General Virology, 3rd Edn. John Wiley & Sons, New York, p4 of 578.  

 

The key words here are UNIT ELEMENT, and INDIVIDUAL: the thing that you see, now, as an organism is merely the current slice in a continuous lineage; the individual evolutionary history denotes the independence of the organism over time. Thus, mitochondria and chloroplasts and nuclei and chromosomes are not organisms, in that together they constitute a continuous lineage, but separately have no possibility of survival, despite their independence before they entered initially symbiotic, and then dependent associations.

The concept of replication is contained within the concepts of individual viruses constituting continuous lineages, and having an evolutionary history.

Thus, given this sort of lateral thinking, viruses become quite respectable as organisms:

• they most definitely replicate,
• their evolution can (within limits) be traced quite effectively, and
• they are independent in terms of not being limited to a single organism as host, or even necessarily to a single species, genus or phylum of host.

Definitions

by EP Rybicki (1999).  An Electronic Introduction to Molecular Virology.  Buglet Electronic Press, Cape Town.

 

Acellular Organisms:
acellular: not composed of cells ( = "bodies of protoplasm made discrete by an enveloping plasma membrane");
Penguin Dictionary of Biology, 9th Edition, 1994

Genome:
The total genetic complement of a virus. This may be composed of RNA or DNA, and be single- or double-stranded. It may also be fragmented.

 

Other Autonomous or Semi-Autonomously Replicating Genomes

There are a number of types of genomes which have some sort of independence from cellular genomes: these include "retrons" or retrotransposable elements, bacterial and fungal (and eukaryotic organelle) plasmids, satellite nucleic acids and satellite viruses which depend on helper viruses for replication, and viroids.   A new class of agents - PRIONS - appear to be "proteinaceous infectious agents" (see also here for an ICTV description, here for some local information and more links).
 

  
Depiction of Prions infecting Neurons: from Russell Kightley Media

Plasmids
Plasmids may share a number of properties with viral genomes - including modes of replication, as in ss circular DNA plasmids and viruses - but are not pathogenic to their host organisms, and are transferred by conjugation between cells rather than by free extracellular particles.

Satellite Nucleic Acids
Certain viruses have associated with them nucleic acids that  are dispensable in that they are not part of the genome, which have no (or very little) sequence similarity with the viral genome, yet depend on the virus for replication, and are encapsidated by the virus.   These are mainly associated with plant viruses and are generally ssRNA, both linear and circular - however, a circular ssDNA satellite of a plant geminivirus has recently been found.

Satellite Viruses
There are also viruses which depend for their replication on HELPER VIRUSES: a good example is tobacco necrosis satellite virus (sTNV), which has a small piece of ssRNA which codes only for a capsid protein, and depends for its replication on the presence of TNV.  Another good example is the hepatitis delta agent with its circular ssRNA genome.  The adeno-associated viruses (AAVs) are also satellite viruses dependent on the linear dsDNA adenoviruses for replication, but which have linear ssDNA genomes and appear to be degenerate or defective parvoviruses.

Viroids
Viroids are small naked circular ssRNA genomes which appear rodlike under the EM, which are capable of causing diseases in plants. They code for nothing but their own structure, and are presumed to replicate by somehow interacting with host RNA polymerase, and to cause pathogenic effects by interfering with host DNA/RNA metabolism and/or transcription. A structurally similar disease agent in humans is the hepatitis B virus-dependent hepatitis delta agent, which additionally codes for a structural protein.

 

Copyright Ed Rybicki,  April, June,1998; March 1999, November 2000

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Retroid Elements and Retroviruses

Retroviridae [ssRNA(+) viruses replicating via a longer-than-genome-length dsDNA intermediate], Hepadnaviridae, caulimoviruses and badnaviruses [family Caulimoviridae, gapped circular dsDNA viruses replicating via longer-than-genome-length RNA intermediates] all share the unlikely attribute of the use of an enzyme complex consisting of a RNA-dependent DNA polymerase/RNAse H in order to replicate. They share this attribute with several retrotransposons, which are eukaryotic transposable cellular elements with striking similarities with retroviruses [such as the yeast Ty element, the mammalian LINE-1 elements, and the Drosophila copia element]; and with retroposons, which are eukaryotic elements which transpose via RNA intermediates, but share no obvious genomic similarity with any viruses other than the reverse transcriptase.

Bacteria such as E coli also have reverse- transcribing transposons -known as retrons - but these are very different to any of the eukaryotic types while preserving similarities in certain of the essential reverse transcriptase sequence motifs.

All of these elements are collectively known as RETROELEMENTS; the fact that the reverse transcriptases of all of them have some amino acid identity suggests a common evolutionary origin.

Several reviewers have pointed out that just such an enzyme as reverse transcriptase would have been necessary for the transition from what is widely believed to have been an RNA world - that is, where all the extant organsisms had RNA genomes - to the present world in which all cellular organisms have DNA genomes.

Viruses with RNA genomes which use RNA-dependent RNA polymerases for their replication may be the only remnants of that pre-DNA era; however, cellular elements and viruses which use reverse transcriptase may share a common origin as cell-derived "modules" coding for a reverse transcriptase, which evolved to become retrons and retroposons and retrotransposons. Addition of structural proteins may have allowed evolution of retroviruses.

The evolution of the DNA retroviruses - Hepadnaviridae, caulimo- and badnaviruses - is more obscure; it appears as though these arose from retrotransposon-like sequences, but this probably occurred near the origin of of these types of element as they are so diverse in sequence and genome organisation.

It is believed that retrotransposons may contribute substantially to the evolution of their hosts. Evidence for this has been obtained by studying human LINE-1s (Long Interspersed Nuclear Elements) - a group of retrotransposable elements which make up approximately 15 % of the human genome. The vast majority of LINE-1s are no longer retrotransposition competent and it is believed that in humans only between 30 and 60 full length LINE-1s are currently active. There is strong evidence from sequences in the sequence databases to suggest that active LINE-1s play an important role in "exon shuffling" (belived to be the major mechanism of macro-evolution whereby entirely new genes are created by reshuffling the components of older genes). The most compelling evidence that LINE-1s do facilitate exon shuffling, however, is the experimental demonstration that they are not only able to move large amounts of non-LINE-1 exonic DNA but also insert this DNA into unrelated expressed genes to obtain chimeras which encode active hybrid gene products.

References:

Chapter 7: "Evolution by transposition" (pp. 172-203) in "Fundamental of Molecular Evolution" by Wen-Hsiung Li and Dan Graur; Sinauer Associates, Inc., Sunderland, Mass., 1991

Moran, J. V., R. J. DeBerardinis, and H. H. Kazazian. 1999. Exon shuffling by L1 Retrotransposition. Science 283:1530-1534.

 

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WHAT ARE VIRIONS?

 

VIRIONS are virus particles: they are the INERT CARRIERS of the genome, and are ASSEMBLED inside cells,  from virus-specified components: they do not GROW, and do not form by DIVISION. 

They may be regarded as the EXTRACELLULAR PHASE of the virus: they are exactly analogous to "spacecraft" in that they take viral genomes from cell to cell, and they protect the genome in inhospitable environments in which the virus cannot replicate.

Excellent examples of virions - and an explanation of their basic structure - may be seen here, at the University of Cape Town's Medical Microbiology Department home page.

For other excellent representations of virus structure, go to the University of Wisconsin's Institute for Molecular Virology, where Jean-Yves Sgro has assembled a gallery of 3-D image reconstructions of different viruses, together with explanations of structure.

 

copyright June 1998 by EP Rybicki unless otherwise stated

Basic Virion Constituents

 

Basically, all virions have:

• genomic nucleic acid: this may be RNA or DNA, ss or ds, and may be all or part of the genome (see also here)
• protein associated with the genome: this may be in the form of a helically-assembling or isometrically-assembling SIMPLE NUCLEOCAPSID only, or may constitute more complex structures with two or more layers of protein, with nucleoprotein innermost.

SOME virions additionally have:

• ENVELOPES, or lipid bilayer MEMBRANES: these always have viral proteins inserted in them (almost always GLYCOPROTEINS); these are often referred to as SPIKES. 
• There is usually an inner layer of protein (usually referred to as MATRIX protein) inside the envelope, usually in contact with the CAPSID protein.   See here for an example (influenza virus, Orthomyxoviridae).

If the virions are simple NUCLEOPROTEINS - that is, contain only nucleic acid and protein - then they are usually  composed ONLY of virus-specified components.  However, certain host components may be "trapped" within virions, such as POLYAMINES: these are polycationic compounds which serve to neutralise charge on the viral nucleic acid as it is packed into the CAPSID, or protein coat.  PAPOVAVIRUSES may in addition encapsidate host histones associated in NUCLEOSOME complexes with virus genomic DNA.

More complex virions - those with membranes, and/or large genomes - may additionally incorporate host membrane proteins in their envelopes: while this may be a side effect of the "budding" process by which viral nucleoprotein complexes acquire envelopes, it may also in some cases be a mechanism for avoidance of host immune systems (eg: hepatitis B virus incorporates host serum albumin into its capsid).

 

 

Helical Nucleocapsids

This is one of the SIMPLEST FORMS of viral capsid: the protein is "wound on" to the viral nucleic acid (generally ssRNA, though M13 and other filamentous phage virions contain circular ssDNA) in a simple HELIX, like a screw (see the diagram for tobacco mosaic virus, below).

courtesy L Stannard

In the case of TMV this is the entire virion: this is also the case for all RODLIKE and FILAMENTOUS virions where no membranes are involved.  This includes all Tobamoviridae, Potyviridae, and Closteroviridae, but NOT Filoviridae, like Ebola virus (see here).

In other cases, filamentous helical nucleocapsids may be enclosed within matrix protein and a membrane studded with spike proteins:   excellent examples of this are PARAMYXOVIRIDAE, images of which can be found here, at Linda Stannard's site.

Isometric Nucleocapsids

These are built up according to simple structural principles, as amply outlined here, and in more detail here.  Put simply, nearly all isometric virions are constructed around a BASIC ICOSAHEDRON, or solid with 20 equilateral trinagles for faces.  It suffices to say that the "quasi-icosahedral" capsid is possibly Nature's most popular means of enclosing viral nucleic acids; they come in many sizes, from tiny T=1 structures (Nanoviruses, eg: banana bunchy top virus; 18 nm diameter) to huge structures such as those of Iridoviridae or Phycodnaviridae (over 200 nm diameter).  A good example of a simple structure is illustrated below in the animated GIF: this shows cowpea chlorotic mottle (CCMV) virion surface structure  (courtesy J-Y Sgro), which is composed of 180 copies of a single coat protein molecule.

The different colours in the picture represent different "positional states" of the capsid protein: subunits around 5-fold rotational axes of symmetry are BLUE, and cluster as PENTAMERS; subunits around 3-fold axes are RED and GREEN to reflect their different 2-fold symmetries; they cluster as HEXAMERS around "local 6-fold axes".

Another recent example - that of turnip yellow mosaic virus (TYMV) - is given here.   This has exactly the same basic structure, with a single type of coat protein subunit, only the pentamer-hexamer clustering is more pronounced

A more complex capsid - that of the common-cold-causing Rhinovirus R16 (family: Picornaviridae), with 60 copies of 4 proteins in a T=3 structure - is shown below (animation modified from one by J-Y Sgro).  This shows a capsid with a cutaway, to reveal internal structure.   BLUE subunits around 5-fold axes are VP1; RED and GREEN are VP3 and VP2 respectively; YELLOW subunits (seen only internally) are VP4.   The VP4 subunits are formed by autocatalytic cleavage of VP0 (into VP2 and VP4) upon binding of a "procapsid" with viral genomic ssRNA.

See here for further details of picornaviruses, here for a scheme showing picornavirus assembly, and here for a scheme outlining polyprotein processing of picornaviruses, and here for material from the Leicester course.

More complex structures may be seen at Linda Stannard's Web site.

 

 

 

Virus Classification

Contents

   

Introduction

A universal system for classifying viruses, and a unified taxonomy, has been established by the International Committee on Taxonomy of Viruses (ICTV) since 1966. The system makes use of a series of ranked taxons, with the:

• - Order (-virales)being the highest currently recognised.
• - then Family (-viridae)
• - Subfamily (-virinae)
• - Genus (-virus)
• - Species ( eg: tobacco mosaic virus)

For example, the Ebola virus from Kikwit is classified as:

- Order Mononegavirales

- Family Filoviridae

- Genus Filovirus

- Species: Ebola virus Zaire

Other Resources:

An excellent resource for virus taxonomy is the International Committee on Virus Taxonomy (ICTV) Database, the ICTVdB, maintained at the Australian National University by Cornelia Büchen-Osmond and Mike Dallwitz.

It is organised as a collection of several pages, accessible as an alphabetical list of virus families (index list mirrored locally); from 1: Adenoviridae, to 80: Viroids, as well as according to nucleic acid content and strandedness and according to general viral host range. Data have been entered in DELTA format, and clicking on any familial or lower taxon gives an immediate output from the database:

for example, clicking on " 03. Arenaviridae ... 03.0.1.1.003 Lassa virus" gives us a full description of the virus and its genome, with links to genome database accessions, references, and pictures if available. An excellent place to start if you know nothing about a given disease agent.

A good illustration of classification of viruses is the Index Virum page.

The ICTV proper has a new Web site too: visit it at the National Center for Biotechnology Information server.

 

The most important taxonomic criteria are:

Host Organism(s)

Particle Morphology

Genome Type

- although a number of other criteria - such as disease symptoms, antigenicity, protein profile, host range, etc. - are important in precise identification, consideration of the above three criteria are sufficient in most cases to allow identification of a virus down to familial if not generic level.

An interactive virus identification key is currently under development at this site: test it and see how the criteria rapidly allow identification.

A key developed by Claude Fauquet and included on the ICTV 6th Report is available at the ICTV Web site at the NCBI; an updated and HTMLised version of it is also available here.

Another site offering a virus identification resource is the Bioinformatics Centre at the University of Pune, India : their resource allows the probabilistic identification of unknown animal viruses, as well as suggesting tests to increase accuracy of identification.

 

Hosts of viruses include all classes of cellular organisms described to date:

General host range

 

  Certain virus families / groupings cross "kingdom" or phylum boundaries.

For example, virus families infecting two kingdoms of organisms are:

• Bunyaviridae (animals and plants)
• Partitiviridae (plants and fungi)
• Reoviridae (animals and plants)
• Rhabdoviridae (animals and plants)
• Phycodnaviridae (protozoa and plants)
• (Picornaviridae) (plants and animals - tentative)
• (Totiviridae) (protozoa / fungi and insects - tentative)

 

Virus families infecting across different phyla (all infecting insects and vertebrates) are:

• Flaviridae
• Iridoviridae
• Parvoviridae
• Poxviridae
• Togaviridae

 

Copyright Ed Rybicki unless otherwise stated, October 1996; November 1997; August 1998

An illustrated tutorial.

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Laboratory diagnosis of viral infections frequently requires the isolation of the virus in cell cultures. Cell monolayers are inoculated with a suitable clinical specimen, and then observed for cytological changes that indicate virus growth.

Cytopathic changes

The term "cytopathic effect" (CPE) is frequently applied to virus-induced cellular changes that are visible by light microscopy. These changes include swelling or shrinkage of cells, the formation of multinucleated giant cells (syncytia), and the production of "inclusions" (made visible by staining) in the nucleus or cytoplasm of the infected cell.

The most efficient way to demonstrate cellular changes is by staining with chromatic dyes.
Cell monolayers are fixed and then exposed to basic and acidic dyes that accentuate the nature and location of the changes.

The use of haemotoxylin (basic dye) and eosin (acidic dye) is often referred to as
H&E staining.

The gross appearance of the cellular changes, and the location and nature of the "inclusions" - i.e. basophilic or eosinophilic - can in many instances be used as a diagnostic criterion to identify the causative virus. These will be illustrated for some of the viruses commonly isolated in cell culture:-

• Adenovirus - example of basophilic intranuclear inclusions
  
• Reovirus - example of eosinophilic intra-cytoplasmic inclusions
  

See also

Syncytia:	The herpes group of viruses:
Measles virus Respiratory syncytial virus Mumps virus	herpes simplex virus human cytomegalovirus varicella zoster virus

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Adenovirus

 

Cells infected with adenovirus have an affinity for haematoxylin (a purplish-blue dye).
Infected cells become rounded and the cell sheet disintegrates. Dark basophilic inclusions within the nuclei represent accumulated viral proteins at the site of virus assembly.

 

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Reovirus

Replication of reovirus particles occurs in the cytoplasm of the cell, and in the final stages of assembly the virus particles bud through the endoplasmic reticulum membrane.
Cytoplasmic sites of accumulated viral protein are stained with eosin (deep pink).

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Go to:
Syncytia:
The herpes group of viruses:

Return to: Medical Microbiology Homepage

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This page was prepared by Linda M Stannard from photographs taken by Diana Hardie,
Department of Medical Microbiology, University of Cape Town.
© Copyright 1996.

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Syncytia

Many enveloped viruses possess a fusion protein in their envelopes. This confers the ability of the virion to fuse with the host cell membrane and thus allow entry of the infectious genomic material into the cell cytoplasm. During replication of the virus, expression of the fusion protein at the cell membrane can result in the fusion of neighbouring cells, and the formation of multi-nucleate cells or syncytia.

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Measles virus

Very large syncytia can be formed during replication of measles virus in cell culture.
An additional distinguishing feature of measles is the presence of distinct eosinophilic inclusions in the nuclei of infected cells.

In the syncytium shown on the right, multiple nuclei are clustered around an eosinophilic cytoplasmic mass that probably represents the Golgi compartments of the fused cells.

Intra-nuclear inclusions are clearly visible.

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Respiratory syncytial virus

As its name implies, respiratory syncytial virus (RSV) causes large syncytia.
Nuclei do not contain inclusions, but pale eosonophilic inclusions can often be seen in the cytoplasm (see below).

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Mumps virus

Mumps virus CPE is indistinguishable from that of RSV (shown above).
However, mumps virus encodes a haemagglutin protein which is incorporated in the virus envelope, and appears at the cell surface from which progeny virions will bud. If erythrocytes (red blood cells) are added to infected cell sheets, they will adhere to the cell surface.
This process is termed haemadsorption
and allows differentiation between mumps and RSV infections.

Haemadsorption of erythrocytes on the surface of cells infected with mumps virus

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Go to:
The herpes group of viruses:
CPE homepage

Return to: Medical Microbiology Homepage

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This page was prepared by Linda M Stannard from photographs taken by Diana Hardie,
Department of Medical Microbiology, University of Cape Town.
© Copyright 1996.

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Entrance, Entertainment and Exit:

The Virus Life Cycle

Viruses have a defined "life cycle" as do any other type of organisms; however, given that they are obligate intracellular parasites, this cycle revolves around:
 

• getting into a host cell

• replicating there, and

• getting out again.

For eighteen years now I have taught this cycle under the heading

"Entrance, Entertainment, and Exit*",

as this is the best mnemonic I know to remind one of the process.  Other courses tend to label these steps as (for example)

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 Virus Entry Into Cells

Replication of Viruses

Assembly and Release of Virions

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These pages link frequently to the University of Leicester Microbiology Dept. Virology 335 course material, mirrored at this site: use the "back" button on your browser to return from a link.

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Back to Contents

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Copyright Ed Rybicki, August 1997, 1998; March 1999 (Unless otherwise stated)

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* = from the Pink Floyd song "The Grand Vizier's Garden Party" (Roger Waters), on the album "Ummagumma".  Which also contains the delightful "Several Species of Small Furry Animal Gathered Together in a Cave and Grooving With a Pict", which title is only a little shorter than the unforgettable Hoagy Carmichaels' "I'm a Cranky Old Yank in my Clanky Old Tank, on the Streets of Yokohama With My Honolulu Mama, Doing Those Beat-O, Street-O, Flat on my Seat-O, Hirohito Blues"...but I digress. 

Click here for Pink Floyd midi files to brighten your browsing.
 
 

  

Virus Entry Into Cells

This 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: these may be defined as:
 

• bacterial (eg: Eubacteria, Archaea)

• animal (eg: all animal cells)

• plant-like (eg: green multicellular plants, algae, fungi).
  

Pictures courtesy of Russell Kightley

Links which explain the basic structures of these kinds of cells may be found at the ESG Biology Hypertextbook Site at MIT and at the WWW Cell Biology Course Site.

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The material here will be divided into the following headings:

1.    BACTERIAL CELL ENTRY

2.    EUKARYOTE CELL ENTRY
    a.    Animal Cell Entry
    b.    Plant Cell Entry
    c.    Fungal Cell Entry

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Back to Contents 

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Copyright Ed Rybicki, August 1997; September 2003
(Unless otherwise stated)

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BACTERIAL CELL ENTRY

Bacterial cell walls are strong and relatively thick, to protect them from osmotic lysis and predation, and to give them shape.  GRAM-POSITIVE cells have a single internal lipid bilayer, and a thick PEPTIDOGLYCAN cell wall.

     
Gram-positive and gram-negative cell walls
(courtesy Russell Kightley)

GRAM-NEGATIVE cells have an internal membrane, a thin peptidoglycan layer, another membrane, and often a polysaccharide-based CAPSULE.  

Bacterial viruses (BACTERIOPHAGES) have therefore to have some means of breaching a quite formidable barrier if they are to enter the cell.  They also generally have SPECIFIC  RECEPTOR SITES on the bacteria, to which SPECIFIC ATTACHMENT PROTEINS bind: these receptor sites may be lipopolysaccharides, cell wall proteins, teichoic acid, or flagellar or pilus proteins.

   

Phage T4 - Enterobacteria phage T4, genus "T4-like Viruses", family Myoviridae, or viruses with 34-170 kbp dsDNA genomes, isometric heads and contractile tails - infects the gram-negative bacterium E coli.  It has one of the more complex entry mechanisms, involving an active injection process.  This is shared by others of the so-called T-even phages of the family Myoviridae.  The process is shown in the still image above left (courtesy Russell Kightley), and in the animated graphic above right (derived from EM images taken by L Stannard, Dept Medical Microbiology, UCT).  

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Click here for an non-animated view of the process if your browser does NOT support GIF animation.

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  The phage tail fibres are the attachment sites; these individually bind the bacterial cell surface - specifically to certain lipopolysaccharides and to the surface outer membrane protein OmpC.  This is REVERSIBLE binding, and is probably due to electrostatic interactions as it is Mg2+ and Ca2+ dependent.  After TAIL FIBRE binding has consolidated, the BASEPLATE then settles down onto the surface and binds firmly to it.  After this occurs, a CONFORMATIONAL CHANGE takes place in baseplate and sheath protein structures, and the TAIL SHEATH CONTRACTS, pushing the TAIL CORE through the cell wall, possibly in an ATP-driven process: this is aided by a lysozyme activity associated with the baseplate assembly.  This is an IRREVERSIBLE process.  DNA is then extruded from the phage head.  This is then used for initial transcription and virus expression.  

Phage lambda - Enterobacteria phage λ, genus "λ-like Viruses", family Siphoviridae, a tailed phage with an isometric head and a 49 kbp dsDNA genome - attaches to the maltose receptor on the surface of the E coli cell.  Although the tail is non-contractile, a DNA injection mechanism similar to that of T-even phages allows entry of DNA into the cell, leaving the capsid behind.

Lambda phage infecting E coli
(courtesy Russell Kightley)

MS2 phage - Enterobacteria phage MS2, genus Levivirus, family Leviviridae - an isometric single-stranded RNA-containing virus infecting E coli - attaches to PILIN  (the building block of PILI) via its single attachment or A PROTEIN.  The A protein is covalently linked to the 5'-end of the genomic RNA; thus, when the pilus is retracted into the cell, the A protein and RNA are pulled with it, leaving the empty capsid outside.

Here is an animated diagram of  a DNA-containing enveloped isometric phage entering a gram-negative bacterium.

Copyright Ed Rybicki, November 1997, June 1998, March 1999; September 2003
(Unless otherwise stated)

Virus Entry Into Cells

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EUKARYOTE CELL ENTRY

a.    Animal Cell Entry

• Animal Cells and Vesicle Transport

• Direct Cell Membrane Fusion

• Entry via Endocytotic Vesicle

• Nuclear Targetting

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Animal Cells and Vesicle Transport

Eukaryote cells do not have cell walls; they have only lipid bilayer cell membranes (=PLASMALEMMA ) with associated  embedded proteins.  They also have a very highly developed INTERNAL MEMBRANE COMPLEX, consisting of endoplasmic reticulum, Golgi apparatus, nuclear membrane, and various specialised vesicles such as LYSOSOMES, which are involved in intracellular digestion.  Eukaryote cells have an intricate system of vesicle transport centred on the Golgi apparatus; this involves export of protein(s) and vesicles from the ER to the Golgi; production of EXPORT vesicles containing proteins from this for fusion with the cell membrane (=EXOCYTOSIS); production of LYSOSOMES  to fuse with ENDOSOMES for digestion of material internalised by RECEPTOR-MEDIATED or non-specific ENDOCYTOSIS (PHAGOCYTOSIS for particulates; PINOCYTOSIS for liquid).  Other CYTOSKELETON-DIRECTED vesicle trafficking involves targetting of vesicles back to the Golgi and to the NUCLEAR MEMBRANE.

The initially-formed ENDOCYTOTIC vesicles resulting from receptor-mediated endocytosis are coated with CLATHRIN, a structural protein which promotes curvature of the membrane at COATED PITS - which become vesicles as the protein continues to assemble and impresses the membrane into a sphere.  Clathrin is recycled back to the cell membrane after endocytosis, either because of interaction of the vesicle with a lysosome, or due to another process.

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Direct Cell Membrane Fusion

Viruses take advantage of these processes in a number of ways, after they have attached to the cell surface via binding to a specific receptor.  The simplest is DIRECT MEMBRANE FUSION, where the virion membrane fuses with the cell membrane, and the virion nucleoprotein complex is delivered into the cell cytoplasm directly.  This is generally a pH-independent process, and requires only that the membrane be fluid (ie: temperature in the physiological range), and generally that some divalent cations be present. 

The entry process for HIV is shown in the graphic (from the Univ Leicester material, copyright Dr AJ Cann).  

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Click here for an non-animated view of the process if your browser does not support GIF animation.

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Here the virion attachment protein - gp120 - (see also here) attaches initially to the CD4 protein on a helper T-cell.  The gp120 undergoes conformational change due to binding, and binds the accessory receptor - CCR-5, a chemokine receptor in this case, although there are others - as well.  gp41 - a cleavage product of a gp160 precursor, and a part of the "spike protein" of the viral membrane - is then able to bind into the cell membrane, via a hydrophobic domain.  A condensation of the gp41 structure - formation of a "6 helix bundle" - causes close juxtaposition of cell and viral membranes, which promotes membrane fusion and nucleoprotein entry into the cell.  An excellent depiction of this process is given on the Roche site.

The entire HIV life cycle is shown here: graphics kindly provided by Russell Kightley.

PARAMYXOVIRIDAE are also able to enter cells in this fashion; in fact, an F or fusion (glyco)protein purified from enveloped virions of Sendai virus is often used to artificially fuse cells in in vitro experiments.

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Entry via Endocytotic Vesicle

Another means of cell entry via membrane interactions is typified by influenza virus: this attaches to cell membranes via its HAEMAGGLUTININ (HA) protein - a trimeric attachment protein - which binds NEURAMINIC ACID residues on cellular glycoproteins.  The process is demonstrated in the accompanying graphic (from the Univ Leicester material, copyright Dr Shaun Heaphy).  

The virion binds reversibly to the cell surface via one or a few attachment proteins. Receptor consolidation results in curvature of the membrane around the particle, and eventual formation of an endocytotic vesicle by a "zipper" process.

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Click here for an non-animated view of the process if your browser does not support GIF animation.

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A cell surface protease cleavage divides the HA into HA1 and HA2: this causes a conformational change which exposes a hydrophobic "fusion domain" on HA2. A further pH-induced structural change (with lysosome fusion, pH drops), allows insertion of part of HA2 into the vesicle membrane.  Another conformational change caused by membrane interaction results in "apposition" of membranes and fusion, allowing escape from the vesicle of the viral nucleoprotein, into the host cell cytoplasm.  This is generally known is pH-dependent entry.

Many enveloped viruses share this mode of entry; so too do many non-enveloped viruses, such as the complex dsDNA adenoviruses (Adenoviridae) and the simpler ssRNA picornaviruses (Picornaviridae).  The former appear to enter via an endocytotic vesicle, then to interact with the vesicle membrane - probably because of structural alterations due to the pH shift - so as to expel a partially-uncoated viral core structure into the cytoplasm.  This is then targetted to the nucleus, where the DNA appears to enter via a nuclear pore.  

Picornaviruses do something similar: that is, the capsid becomes rearranged as a result of pH-induced structural transitions, whereby the VP4 internal protein is lost and the surface becomes more lipophilic, and interacts with a vesicle membrane so as to allow exit of the RNA into the cytoplasm.  A more complex process may also occur which allows RNA to enter the cell from externally-bound virions.

These mechanisms are summarised in this diagram: click here to link to source page.

Reoviridae - isometric dsRNA non-enveloped viruses with up to 12 genome components - enter by endocytosis, are partially uncoated in lysosomes, and then core particles enter the cytoplasm by an unknown process, perhaps similar to what occurs with adenoviruses.  Core particles then function as virus transcription "factories"
 

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Nuclear Targetting

A basic requirement in the infection of Eukarya would be that DNA genomes end up in the nucleus - except for Pox-, Phyco- and some Baculoviridae - and that RNA genomes end up in the cytoplasm - except for myxoviruses - as these are the sites where the respective viruses may be expected to replicate their genomes.  DNA is its own nuclear targetting signal in that naked DNA is seen to be moved into cell nuclei if introduced into cells; however, most DNA viruses which require a nuclear replication stage seem to have some specific means of targetting partially-disassembled virions to the cell nucleus. Nucleoproteins with nuclear localisation signals (NLS) are very often involved.

Adenoviruses  - large naked isometric virions with linear dsDNA of 30-38kbp - appear to be transported by means of the hexon protein of the partially degraded naked capsid that is released into the cytoplasm, interacting with microtubules.  This reaches a nuclear pore and allows escape of viral DNA plus certain viral polypeptides into the nucleus.

Parvoviruses - small naked isometric viruses with linear ~5 kb ssDNA genomes - enter host cells by receptor-mediated endocytosis, escape from endosomal vesicles to the cytoplasm, and then replicate in the nucleus.  Canine parvovirus virions at least use the motor protein dynein to be transported along microtubules to the nucleus.

Herpesviruses enter by fusion with the cell membrane, and the core particles migrate to nuclear pores, and release DNA there.

Poxviruses - which have "intracellular" single-enveloped and "extracellular" double-enveloped forms - may enter by direct cell fusion (pH-independent) or lysosomal vesicle fusion (pH-dependent and inhibitable by lysomotropic agents), with the latter perhaps predominating.  Once core virions are in the cytoplasm, they uncoat further to expose a nucleoprotein complex which is first transcriptionally and later, replicationally, active.

Copyright Ed Rybicki, November 1997, April 1998, June 1998, March 1999, February 2001; September 2003
(Unless otherwise stated)

Virus Entry Into Cells

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EUKARYOTE CELL ENTRY

b.    Plant Cell Entry

c.    Fungal Cell Entry

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Plant Cell Entry

Plant cells, while superficially similar to animal cells in basic construction as far as all organelles except chloroplasts and often extensive vacuoles are concerned (see here), have one large and fundamental difference to animal cells, which profoundly affects the way in which they are infected by viruses, and how viruses move between them.

This is their possession of thick, rigid, cellulose-based cell walls. 

courtesy of Russell Kightley

Every cell is separated from every other cell by cell walls, whose dimensions are far larger than the size of the average virion (ie: >1 micron, cf. TMV = 300 nm).  This means that plant cells are effectively inaccessible to viruses, even given mechanisms of injection similar to the T-even phages.  Live plant cells interconnect only via specific discontinuities in the cellulose walls: the most numerous of these are plasmodesmata, which are complex structures filled with membrane-derived processes continuous with the endoplasmic reticulum. These are "gated" intercellular channels, which limit the passage of molecules between cells, and certainly do not admit particles as large as virions. Plant viruses have therefore evolved specific movement functions, mediated by one or more virus-specified proteins, which interact with the plasmosdesmatal machinery so as to increase the "pore" size, and allow specific transport of viral nucleoprotein complexes.  All plant-infecting viruses possess one or more movement-related protein (MP) genes: these are very varied, although there are distinct groups of them, and they appear to derive from host plant genes for chaperonins and plasmodesmata-associated proteins.

Plant viruses, therefore - which are almost overwhelmingly ssRNA +ve sense and non-enveloped - do not appear to specifically interact with host cell membranes or cell walls, as do bacterial and animal viruses; even when the plant-infecting virus also infects an animal (eg: plant rhabdoviruses and bunyaviruses) and presumably behaves normally in the other host, and even though apparently plant cells are capable of phagocytosis / endocytosis.  The mechanisms employed to enter cells rather appear to be passive carriage through breaches in the cell wall in the first instance, followed by later cell-to-cell spread in a plant by means of specifically-evolved "movement" functions, and perhaps spread via conductive tissue as whole virions.

The "passive carriage" referred to above could mean:

• 1) a purely mechanical injury that breaches the cell wall and transiently breaches the plasma membrane of
  underlying cells;
• 2) similar gross injury due to the mouthparts of a herbivorous arthropod, such as a beetle;
• 3) injection directly into cells through the piercing mouthparts of sap-sucking insects or nematodes;
• 4) carriage into plant tissue on or in association with cells of a fungal parasite;
• 5) vertical transmission through infected seed or by vegetative propagation;
• 6) transmission via pollen; and
• 7) grafting of infected tissue onto healthy tissue.

ONE EXCEPTION to the above rule are some of the PHYCODNAVIRIDAE, which have large (>300 kb) dsDNA genomes, and very large (130 - 200 nm), complex virions.  Exemplars which infect algae appear to have specific enzymes at the surface of virions which may degrade the CHITIN cell wall of the alga , to allow interaction of the particle with the cell membrane directly. Some of these viruses also appear to be able to "inject" their DNA, much as phages do, and to enter a lysogenic state.

The mode of transmission of viruses affects their concentration and localisation in plants. For example, mechanically transmitted viruses (eg: Bromoviridae, Tobamoviruses) tend to reach very high concentrations in most tissues of a plant (up to 4g / kg plant): this is necessary for survival, as it guarantees that a large number of virions will be present for onward transmission by whatever non-specific means presents itself. Viruses which are introduced into plants via insect vectors with piercing mouthparts, on the other hand, tend to be limited in their multiplication to phloem elements, which are preferred target tissues for insect feeding. Consequently, these viruses (eg: Luteoviruses, Geminiviridae) reach only very low concentrations (mg / kg) in whole plants.

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c.    Fungal Cell Entry

There are certain superficial similarities between plants and FUNGI with respect to the cell wall; however, in the latter case, cell walls are composed of CHITIN, a different complex polysaccharide, and fungal hyphae are often effectively "tubes" with no cross-walls.

No fungal viruses appear to have any specific mechanisms for gaining entry to fungal cells; indeed, it is extremely difficult to demonstrate the infectivity of virus-like particles, and it is only since the advent of the GENE GUN or biolistic transformation apparatus, that many viruses have been shown to be infectious at all - by being "shot" into fungal cells adsorbed onto metal particles.

It is probable that most fungal viruses - like the plant-infecting CRYPTOVIRUS genus of the mainly fungus-infecting Partitiviridae - are only transmitted by "grafting", or the physical connection of infected to healthy cells by anastomosis.  Thus, fungal mating is a good means of transmission, as it results in the mixing of cell contents of different hyphae. 

Copyright Ed Rybicki, August 1997, October 2000; September 2003
(Unless otherwise stated)

 

Genome Diversity

 
 

* = viroids only

 

Viruses are the only organisms on this planet to still have RNA as their sole genetic material. They are also the only autonomously replicating organisms to have single-stranded DNA. The range of virus genomes as found in virions encompasses:

• single-component dsDNA, linear or circular (occasionally circularly permuted linear);
• single, double or multi-component circular ssDNA;
• single-component linear ssDNA;
• single or multi-component dsRNA;
• single or multiple component ssRNA genomes which may be
• totally "positive"(or messenger) polarity,
• totally "negative" (or anti-messenger) polarity, or
• partially positive negative-sense;
• "diploid" positive-sense ssRNA genomes which replicate via reverse transcription to and transcription from longer-than-genome-length dsDNA, and
• nicked and/or partially dsDNAs which replicate via transcription to and reverse transcription from longer-than-genome-length positive-sense ssRNA.

In contrast, prokaryotes have mainly single-component circular (occasionally multiple) or linear dsDNA (Streptomyces, Helicobacter) while all eukaryotes have multi-component linear dsDNA, and all the genomes replicate via the classic semi-conservative route.

Viral genome types have inspired a classification based on what is found in virions, coupled with their replication strategy.  This is the Baltimore Classification, and will be discussed in a broader treatment of  Virus Replication.

An overview of the taxa Recognized in the Universal System of Virus Taxonomy in terms of their genome content may be found here.

Copyright Ed Rybicki, August 1997, 1998; March 1999

 

Genomic Replication Strategies of Viruses

The old terms "eclipse phase" or "latent period" describe that part of a virus life cycle when no infectious virus can be extracted from cells which had just been exposed to infectious virions: a good illustration of the concept in terms of a virus assay experiment is shown here.

What happens once a virus is uncoated, or partially uncoated, depends largely upon what sort of virus it is.  The Baltimore Classification of viruses by their genome types and replication strategies makes it fairly easy to predict the broad sort of strategy that a virus with a given genome will employ in order to get replicated.  This classification was originally devised by David Baltimore; it originally only had six categories, but the discovery of "DNA retroviruses" in the 1980s necessitated a new Class VII.
 

To understand the classification, one must understand how cells replicate their genomes, and express mRNAs, and  proteins.  This is conveniently explained by the "Watson-Crick Central Dogma", which states that:

INFORMATION FLOW IN CELLS GOES FROM:

·         DNA TO DNA (REPLICATION)

·         DNA TO RNA (TRANSCRIPTION)

·         AND RNA TO PROTEIN (TRANSLATION)

This may conveniently be described in terms of a diagram showing "information flow":

By contrast, viral replication is far more complicated in terms of information flow:

The Baltimore Classification of Viruses According to Their Genome Types and Their Replication Strategies

The classes are:

See also section on "RNA Virus Replication"

 

 

Copyright Ed Rybicki, August 1997, 1998, March 1999, October 2000
(Unless otherwise stated)

Class I:

Steps in Replication

1. Primary transcription by host enzymes

2. Translation of early (=regulatory) proteins

3. Viral genomic DNA replication (usually by host enzymes)

4. Late transcription (usually mediated by viral proteins)

5. Synthesis of late (=structural) proteins

6. Assembly of structural protein and DNA into virions

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Class II:

Steps in Replication

1. Conversion into dsDNA (=host repair process?)

2. Early transcription (by host enzymes)

3. Translation of (regulatory) protein and "rolling circle" ssDNA replication

4. Late transcription (usually mediated by viral proteins)

5. Synthesis of late (=structural) proteins

6. "Sequestering" of viral genomic ssDNA

7. Assembly into virions

 

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Class III:

Steps in Replication

1. Primary transcription in virion core in cytoplasm by viral RDRP, and export of (+)sense RNA to cytoplasm

2. Translation of (+)sense RNA, accumulation of viral proteins

3. Assembly of (+)sense RNA and viral proteins into immature virions

4. Transcription of (+)sense RNA into dsRNA in virions by viral RDRP

5. Secondary transcription of dsRNA

6. Final assembly / maturation of virions

 

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Class IV:

Steps in Replication

1. Translation of virion RNA as mRNA (early products=RDRP)

2. Synthesis of (-)sense RNA on (+)sense template by RDRP (=formation of replicative complex, RC)

3. Synthesis of (+)sense RNA, mRNA and (-)sense RNA

4. Translation of (+)sense and mRNA, synthesis of structural protein (which biases RC to produce (+)sense RNA?)

5. Assembly of structural protein and (+)sense RNA and maturation of virions

 

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Class V:

Steps in Replication

1. Primary transcription of virion (-)sense RNA by RDRP in virion core in cytoplasm, production (mainly) mRNA and (+)sense RNA, formation replicative complex (RC)

2. Translation mRNAs, accumulation of products

3. Virion proteins interact with RC, bias it towards production of full-length (+)sense RNA and therefore of genomic (-)sense RNA

4. Secondary transcription from progeny (-)sense RNA, translation, accumulation structural proteins

5. Nucleocapsid assembly and maturation, budding of nucleocapsid through host membrane containing viral envelope proteins

 

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Class VI:

Steps in Replication

1. Reverse transcription in cytoplasm, using tRNA primer, of virion ss(+)RNA by virion-associated reverse transcriptase (RT), into intermediate RNA/DNA complex

2. Conversion of RNA/DNA complex into linear and circular proviral dsDNA forms with long terminal repeats (LTRs) by RT; import into nucleus

3. Integration of linear proviral DNA into host cell DNA, by means of integrase function of RT

4. Replication and trasncription as for host DNA, using host enzymes

5. Modification of transcription by (early) viral products (bias to production of genome-length (+)sense RNA?)

6. Translation, accumulation of (late) structural protein, assembly with (+)sense genomic RNA into viral nucleoprotein, budding through membrane containing viral envelope (glyco)proteins

 

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Class VII:

Steps in Replication

1. Entry of viral DNA into nucleus, conversion of "gapped" genomic DNA into cccDNA by host repair synthesis

2. Transcription by host RNA pol into mRNA(s) and longer-than-genome-length "genomic" (+)sense RNA

3. Translation of mRNA and (+)sense RNA in cytoplasm, accumulation of viral products

4. Interaction of viral proteins with (+)sense RNA, assembly of provirions, and reverse transcription of RNA inside virions by viral RT to RNA/DNA complex

5. Conversion of RNA/DNA complex to circular, gapped dsDNA by virion RT

6. Final virion maturation, arrest of further DNA polymerase activity in virion (budding for hepadnaviruses)

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