WHAT IS A VIRUS

Infectious Intracellular RNA or DNA genome, not both Protein coat* May of may not have lipid envelope May have broad or narrow host range Replication involves eclipse (breaking apart of virus particles) and reassembly

Viruses are submicroscopic pathogens

Viruses may be simple or complex

Genome sizes 0.3 - 300 kb; average sizes vary with host organism types May have single-stranded or doublestranded RNA or DNA genome If RNA, may be + or sense May have one or many proteins in particles May or may not have lipid envelope

Typical +sense RNA virus replication cycle

1. Virus enters host cell 2. RNA released; translates

3. Replication in cytoplasm 4. New virus is assembled

From Cann 1997

Composition of viruses infecting different kingdoms

No rules about virus families that may or may not be present in a given kingdom Some types of viruses are found more in some kingdoms than in others
Many plant viruses contain ssRNA genomes Many fungal viruses contain dsRNA genomes Many bacterial viruses contain dsDNA genomes

Host properties determine the types of viruses that tend to be found in members of a biological kingdom Complexity of viruses in different families varies considerably

Types of viral genomes

double-stranded (ds) DNA
Rarely segmented Often large

single-stranded (ss) DNA

Rarely large Less common than dsDNA

ssRNA, negative sense

Often found in viruses with broad host ranges

ssRNA, positive sense

Most common overall

dsRNA
Often segmented Particle structure often critical

Virus types, by nucleic acid

DNA ss ds ss RNA ds

Families Species Host type Vertebrate Invertebrate Plant Fungus Bacteria

env naked env naked env naked env naked 0 5 9 12 9 14 2 5 0 100 200 300 200 600 10 300 + + ++ + ++ ++ + ++ + + +++ ++ ++ + + ++ ++ +++ + + + + ++ ++ + +++ -

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

From 7th Report of the ICTV (Academic Press, 2000)

VIRUS STRUCTURE
Basic rules of virus architecture, structure, and assembly are the same for all families Some structures are more complex than others The capsid (coat) protein is the basic unit of structure; functions that may be fulfilled by the capsid protein are to:
Protect viral nucleic acid Interact specifically with the viral nucleic acid for packaging Interact with vector for specific transmission Interact with host receptors for entry to cell Allow for release of nucleic acid upon entry into new cell Assist in processes of gene regulation

Nucleoprotein has two basic structure types: Virus structure is studied by:
Transmission electron microscopy (EM) Cryo-EM one of the most powerful methods currently available X-Ray diffraction Neutron scattering Primary sequence analysis Protease and footprinting Site-directed mutagenesis

Atomic Resolution Microscope at UC Berkeley The Atomic Resolution Microscope is specifically designed for performance in the high resolution imaging mode with a point-to-point resolution of 1.5.

Typical modern transmission EM: This JEOL Transmission Electron Microscope, similar to the one we use at Rutgers, is housed at Colorado State University

Typical transmission electron micrograph of negatively stained, purified virus preparation (calicivirus)
Note that heavy metal stain penetrates into spaces, resulting in electronopaque areas against electrontransparent protein background in particles

Cryo-electron microscopy
(excerpted from Norman Olson, Purdue U.)
Cryo-EM is TEM in vitreous ice
Vitreous ice is water frozen to -140 C in less than 10-4 sec Vitreous ice state must be maintained in microscope

Advantages of Cryo-EM
Preserves native structure of sample Reduces electron beam damage Allows examination of large, complex macromolecules

Disadvantages of Cryo-EM
Technically difficult Samples are sensitive to beam damage Images have low contrast

From N. Olson web site

From N. Olson web site

From N. Olson web site

From N. Olson web site

From N. Olson web site

From N. Olson web site

Nucleoprotein has two basic structure types:

HELICAL: Rod shaped, varying widths and specific architectures; no theoretical limit to the amount of nucleic acid that can be packaged CUBIC (Icosahedral): Spherical, amount of nucleic acid that can be packaged is limited by the of the particle

Principles of virus structure

Nucleoprotein must be stable but dissociatable Capsid is held together by non-covalent, reversible bonds: hydrophobic, salt, hydrogen bonds Capsid is a polymer of identical subunits Terms:
Capsid = protein coat Capsomere = subunit clusters visible in EM Structural unit = protein subunit Nucleocapsid = nucleic acid + protein Virion = virus particle Fold only one way, and robustly Vary in size, generally 50-350 aa residues Have identifiable domains Can be described topologically; similar topological features do not imply evolutionary relationships

Capsid proteins are compactly folded proteins which:

Helical symmetry
Tobacco mosaic virus is typical, well-studied example Each particle contains only a single molecule of RNA (6395 nucleotide residues) and 2130 copies of the coat protein subunit (158 amino acid residues; 17.3 kilodaltons)
3 nt/subunit 16.33 subunits/turn 49 subunits/3 turns

TMV protein subunits + nucleic acid will self-assemble in vitro in an energy-independent fashion Self-assembly also occurs in the absence of RNA TMV rod is 18 nanometers (nm) X 300 nm

Cubic (icosahedral) symmetry

TBSV icosahedron is 35.4 nm in diameter

Tomato bushy stunt virus is typical, wellstudied example Each particle contains only a single molecule of RNA (4800 nt) and T= 3 Lattice 180 copies of the coat C protein subunit (387 aa; 41 kd) Viruses similar to TBSV will self-assemble in N vitro from protein subunits + nucleic acid Protein Subunits Capsomeres in an energyFrom http://mmtsb.scripps.edu/viper/viper.html independent fashion

From N. Olson web site

CLASSIFICATION & NOMENCLATURE of VIRUSES A large number of morphologically and physico-chemically distinct types of viruses that infect virtually all classes of living organisms have been described. Generating some order from the chaos that existed with respect to the naming and cataloguing of viruses became the task of the International Committee on the Nomenclature of Viruses (ICNV), which first met in 1966. In keeping with the spirit of taxonomy, the committee changed its name to the International Committee on the Taxonomy of Viruses in 1973, which is what it is today. The 7th Report of the ICTV was published in Spring, 2000. It contained three orders, 56 families, 9 subfamilies, 233 genera, and 1550 virus species. Descriptions of satellite viruses, viroids, prions, and retrotransposons are included. The concept of classifying viruses on the basis of virus properties as opposed to host affinities or biological effects has integrated the field and allowed for a sensible approach to the study of comparative virology. A centralized repository of virus information, the ICTV database (ICTVdb) is under construction and nearly completed. It includes information gathered from several large databases from around the world. The availability of such information has facilitated the job of accurate identification and diagnosis of new and important virus diseases. The characterization of viruses and their assignment to specific groups and families has had a major impact on what properties virologists must examine in order to properly identify and classify the causal agent of a specific virus disease. Our understanding of the interrelationships among different viruses, genera, and families continues to increase, much through comparative sequence analysis. We therefore continue to gain more confidence in the system we use to classify viruses. Until recently, many viruses were not associated with specific taxa and most plant viruses were categorized into groups rather than families or genera. As more sequence data have accumulated, more viruses have been placed in newly described or existing taxa, and the group designation has given way to categorization into families or genera of existing families. Some viruses are still are not associated with named families, but with the current taxonomic framework solidly in place, this number is being reduced with each report of the ICTV.

Virus Orthography: Orthography is how something is written. Virus orthography changed in 1999, when the ICTV decided that virus names should be italicized, just as true Latin binomials are. This is a matter of considerable controversy among virologists, since virus nomenclature and orthography do not lend themselves to the binomial structure otherwise. Section IX of the ICTV Code reads as follows: 3.39 In formal taxonomic usage, the accepted names of virus Orders, Families, Subfamilies, and Genera are printed in italics and the first letter capitalized. 3.40 Species names are printed in italics and have the first letter of the first word capitalized. Other words are not capitalized, unless they are proper nouns or parts of proper nouns. 3.41 In formal usage, the name of the taxon shall precede the taxonomic unit.
Further Reading *Koonin, E.V., and Dolja, V.V. 1993. Evolution and taxonomy of positive-strand RNA viruses: Implications of comparative analysis of amino acid sequences. Critical Reviews in Biochemistry and Molecular Biology 28:375-430. (This article puts RNA virus evolution in proper perspective.) *Mayo, M. A. and Horzinek, M. C. 1998. A revised version of the international code of virus classification and nomenclature. Archives of Virology 143: 1645-1654.(Describes recent revisions to virus taxonomy, nomenclature, and orthography.) Van Regenmortel et al. 2000. Virus Taxonomy: Seventh Report of the International Committee for the Taxonomy of Viruses. (Most up-to-date, full-scale, official reference on virus taxonomy. A copy of this volume is available in my office [BIH].)

Classification, nomenclature, and orthography

What is the purpose of classification?
To make order To be able to communicate with each other To assemble like members with each other

Various virus classification schemes have been used

Host & symptoms have been important considerations Particle morphology was important after EM developed Physico-chemical properties became important later
Sedimentation coefficient and density of particles Protein compositions and sizes Nucleic acid types, numbers, and sizes

Sequence analysis confirmed most relationships that were inferred otherwise, and revealed new ones
Molecular phylogeny now a primary tool for classifying viruses Complete genomes can be analyzed relatively quickly

Classification, nomenclature, and orthography

Latin binomials were proposed first by Holmes in 1939 Various other schemes proposed between 1940 and 1966 1966 the International Committee for the Nomenclature of Viruses formed; met in 1970 Changed to the International Committee for the Taxonomy of Viruses in 1973 7th Report of the ICTV was published in 2000
56 families, 9 subfamilies, 233 genera, and 1550 virus species Includes retrotransposons, satellites, viroids, prions

Rules of orthography changed in 1999 to require italics, but no true binomial only modified binomial e.g.
Family Reoviridae
Genus Orbivirus
Species Bluetongue virus (24 named strains: BTV-1 to BTV-24)

Criteria for species demarcation vary for different families