Virology

Classification of Virus
By phenotypic characteristics,
Morphology Nucleic acid type Mode of replication Host organisms Type of disease they cause.

Two schemes for the classification of viruses

ICTV system (International Committee of Taxonomy of Viruses) Baltimore classification system

 ICTV:
According to ICTV Viral classification starts at the level of order Order (-virales) Family (-viridae) Subfamily (-virinae) Genus (-virus) Species (generally from the disease)

 Currently 6 orders, 87 families, 19 subfamilies, 349 genera, and 2,284 species of viruses have been defined
Caudovirales : (tailed dsDNA) Herpesvirales : (large eukaryotic dsDNA viruses) Mononegavirales : (nonsegmented (-) strand ssRNA plant and animal viruses) Nidovirales : [(+) strand ssRNA viruses with vertebrate hosts] Picornavirales : (small (+) strand ssRNA viruses that infect a variety of plant, insect and animal host) Tymovirales : (mono partite (+) ssRNA viruses that infect plants)

Baltimore classification
Baltimore classification is a classification system that places viruses into one of seven groups designated by Roman numerals depending on a combination of their nucleic acid, strandedness, Sense, and method of replication. I: dsDNA viruses (e.g. Adenoviruses, Herpesviruses, Poxviruses) II: ssDNA viruses (+)sense DNA (e.g. Parvoviruses) III: dsRNA viruses (e.g. Reoviruses) IV: (+)ssRNA viruses (+)sense RNA (e.g. Picornaviruses, Togaviruses) V: ()ssRNA viruses ()sense RNA (e.g. Orthomyxoviruses, Rhabdoviruses) VI: ssRNA-RT viruses (+)sense RNA with DNA intermediate in lifecycle (e.g. Retroviruses) VII: dsDNA-RT viruses (e.g. Hepadnaviruses)

Cultivation of viruses
Embryonated Egg Live animals Bacteria Live plant tissue Tissue culture :
Organ culture, explant culture, cell culture

Plant cell culture Protoplast

Embryonated Egg

Shell surface is first disinfected with iodine and penetrated with a small sterile drill. After inoculation, the drill hole is sealed with gelatin and the egg incubated (8-11 days). The infection may produce a local tissue lesion known as a pock, whose appearance often is characteristic of the virus.

Bacteria
Bacteriophages are cultivated in either broth or agar cultures of actively growing bacterial cells. Agar cultures are prepared by pouring mixture of bacteriophage sample with cool, liquid agar and a suitable bacterial culture in a petri dish. Bacteria forms an opaque layer or lawn. Virus infects an adjacent cell and reproduces. Eventually, bacterial lysis generates a plaque or clearing in the lawn

Animal cell culture

A layer of animal cells in a petri dish is covered with a virus inoculum, and the viruses are allowed time to settle and attach to the cells. The cells are then covered with a thin layer of agar to limit virion spread so that only adjacent cells are infected by newly produced virions. Plaques of dead cells formed indicate the growth of virus Many times virus will not form plaquebut, causes microscopic or macroscopic degenerative changes or abnormalities in host cells and in tissues called as cytopathic effects

 Viruses also can be grown in whole plants. Leaves are mechanically inoculated when rubbed with a mixture of viruses and an abrasive such as carborundum. When the cell walls are broken by the abrasive, the viruses directly contact the plasma membrane and infect the exposed host cells. A localized necrotic lesion often develops due to the rapid death of cells in

TMV

Tobacco mosaic virus (TMV)

First virus to be discovered Helical structre + ssRNA virus (6400 bases long) Capsid made up of 2130 molecules of coat protein The coat protein self-assembles into the rod like helical structure (16.3 proteins per helix turn) around the RNA which forms a hairpin loop structure The protein monomer consists of 158 amino acids which are assembled into four main alpha-helices, which are joined by a prominent loop proximal to the axis of the virion. Virions are ~300 nm in length and ~18 nm in diameter. Negatively stained electron microphotographs show a distinct inner channel of ~4 nm. The RNA is located at a radius of ~6 nm and is protected from the action of cellular enzymes by the coat protein.

STRUCTURAL FEATURES OF TMV

The protein coat is composed of about 2130 copies of a small protein
CAPSID Helical symmetry 300 nm RNA genome 18 nm 2 nm

NC protein
Images:P. Sforza, 2001 www.ppws.vt.edu/~sforza/tmv/tmv.html

 Genome:
RNA 6395 nucleotides Contains 4 genes
MTH (Methyl tranferase & RNA Helicase) RNP (RNA depended RNA polymerase) MP (movement protein) CP (Coat protein)
6,395 nt RNP Capsid CP 183K 126 K 30 K Movement MP MT-H 17.6 K

5 cap

tRNAhis

TMV Life cycle

a) Virus entry trough abrasions on plant tissue. Inside cell associates with ER b) spontaneous release of few capsid (CP) subunits 5' end of genome is uncovered c) Host ribosome attaches to viral RNA, moves down displacing more CP units
/

5 cap

Host Rb

Modification of a diagram from: L. Stannard, Department of Medical Microbiology, U. of Cape Town

TMV Life cycle

(cont.)

d) Ribosome meets start codon, translates first two proteins (126K ,183 K) while uncoating continues co-traslational disassembly e) 126 K ( MEH) & 183 K ( RNP) use viral RNA as template to make full length complementary neg. strand RNA /

Modification of a diagram from: L. Stannard, Department of Medical Microbiology, U. of Cape Town

TMV Life cycle

(cont.)

RNP

+ Strand (genome)

Neg. strand

f) Neg. RNA strand used by viral replicase (RNP/MEH) as template for +RNA
promoters

g) Also, neg. RNA strand has internal promoters used by replicase to make mRNA for 30K protein (MP) and 17.5 K (CP)

Transcription by RdRp

MP mRNA CP mRNA

TMV Life cycle

(cont.)

h) MP combines with viral +RNA to move it into new plant cells through plasmodesmata

http:www.sunysb.edu/biochem/BIOCHEM/facultypages/citovsky/TMV-TransportModel-III2.jpg

Adenovirus
Adenoviruses are (90100 nm), nonenveloped icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome (26-45 kbp). Capsid:
Capsid proteins (Hexon, Penton and fibre) Minor proteins Core proteins

 Hexon: (967 aa)

Trimer, on the 20 facets of the icosahedral capsid, There are three tower regions that are presented to the exterior and one loop and two eight stranded jelly like rod in the interior There are 240 hexons in the capsid

Penton: (471 aa)

Consists of two domains

Fibre: (582aa)
The fibre has three distinct regions: tail, shaft and knob Bound to penton non-covalently by its N terminus A sequence near the N terminus and a peptide sequence that bonds to the penton are found to be highly conserved between serotypes

 Events in infection:
Through (i) CAR receptor (coxsackie adenovirus receptor) and (ii) CD46 receptor Entry into the host cell is initiated by the knob domain followed by interaction of penton with integrin resulting in internalization of virus by endocytosis through clatherin-coated pits . Once the virus is inside the host cell, the endosome acidifiesand capsid disassociate. With the help of cellular microtubules, the virus is transported to the nuclear pore complex, whereby the adenovirus particle disassembles and vDNA enter into the nucleus. After this the DNA associates with histone molecules. Thus, viral gene expression can occur and new virus particles can be generated.

The adenovirus life cycle is separated by the DNA replication process into two phases: an early and a late phase. In both phases, a primary transcript that is alternatively spliced to generate monocistronic mRNAs compatible with the hosts ribosome is generated, allowing for the products to be translated The early genes are responsible for expressing mainly non-structural, regulatory proteins. The goal of these proteins is threefold:
to alter the expression of host proteins that are necessary for DNA synthesis; to activate other virus genes (such as the virus-encoded DNA polymerase) to avoid premature death of the infected cell by the host-immune defenses

Once the early genes have liberated adequate virus proteins, replication machinery, and replication substrates, replication of the adenovirus genome can occur. A terminal protein that is covalently bound to the 5 end of the adenovirus genome acts as a primer for replication. The late phase of the adenovirus lifecycle is focused on producing sufficient quantities of structural protein to pack all the genetic material produced by DNA replication. Once the viral components have successfully been replicated, the virus is assembled into its protein shells and released from the cell as a result of virally induced cell lysis.

Life cycle of animal virus

Attachment Penetration Replication of genome and synthesis of capsid Assembly Release

Poliovirus
Poliovirus, causes Poliomyelitis Icoshedral, ssRNA + (7440bp). At 5 end is protein called as VPg Capsid is made up of 60 capsomere unit (made up of 4 distinct proteins)

Infection cycle
Attachment and penetration:
Receptor CD155 (PVR) Penetration through endosomes

Translation of RNA
Monocistric RNA Produce a single protein called polyprotein (2200aa) Polyprotein undergoes self cleavage to produce 20 smaller proteins
Structural proteins VPg proteins RNA polymerase Viral protease

 Replication:
RNA polymerase act as replicase Synthesis strand from +strand -strand then act as a template for synthesis of +strands It is followed by assembly of the virion and release by lysis

 Cytopathic effect:

Influenza virus
There are three types of influenza viruses: A, B, and C. Mostly infect human and birds (avian Influenza) Humans can be infected with influenza types A, B, and C viruses Most important serotypes are H1N1, H3N2

Structure
Enveloped ss RNA virus 6-9 nm dia. 600 nm long RNA is segmented ; in Influenza A, it is 8 linear segments (890 2341 bases) Nucleocapsid is helical, but virus is polymorphic (no defined shape) due to the envelop Several surface glycoproteins are present
Hemagglutinin (HA) Neuraminidase (NA). M2 proteins

Capsid is made up of matrix protein M1 RNA depended RNA polymerases (covert RNA to + RNA) RNA endonucleases

Influenza Antigenic shift and drift

Antigenic shift refers to an abrupt, major change to produce a novel influenza A virus subtype in humans that was not currently circulating among people Antigenic drift refers to small, gradual changes that occur through point mutations in the two genes that produce hemagglutinin, and neuraminidase. Antigenic drift produces new virus strains that may not be recognized by antibodies to earlier influenza strains.

Pox virus

 Very large viruses Eg. Vaccinia (400 X 240 X 200 nm)

Retro viruses
Enveloped 80110 nm diameter Genome: ss +RNA, 910 kb Contains reverse transcriptase

Viral genome
The virion contains two copies of the RNA genome, present as a dimer, formed by base pairing between complementary sequences (kissing loop complex.) Viral genome contains few molecules of host tRNA sequence bound to each copy of the virus through base pairing. The sequence in the virus RNA that binds a tRNA is known as the primer binding site (PBS) which is highly specific for each retrovirus.

Viral capsid (HIV)

Capsid is cone shaped 4060 nm dia. At the wide end 20 nm at the narrow end. Virus with two or more capsids have been reported.

Proteins CA capsid IN integrase MA matrix NC nucleocapsid PR protease RH ribonuclease H RT reverse transcriptase SU surface glycoprotein (gp120 ) TM transmembrane glycoprotein (gp41)

9.3 kb in length Genes gag group-specific antigen pol polymerase env envelope auxiliary genes: controlling virus gene expression, transporting virus components within the cell and modifying the hosts immune response.

HIV genome

Replication
Attachment and entry
gp120 binds to CD4 receptor and a chemokine coreceptor (CCR5 or CXCR) re-arrangement of gp41, which proceeds to fuse the membranes of the virion and the cell The contents of the virion envelope are released into the cytoplasm and develop into the reverse transcription complex, which contains the MA, Vpr, RT and IN proteins, as well as the virus genome

 Reverse transcription and transport to the nucleus

The reverse transcription complex associates rapidly with microtubules Reverse transcription is primed by tRNAlys-3 Synthesis of both the () DNA and the (+) DNA begins at the 3OH of a primer RNA. The primer for synthesis of the () DNA is the tRNA bound to the genome, while the primer for synthesis of the (+) DNA is a polypurine tract (PPT) in the virus genome.

 After reverse transcription has been completed, the pre-integration complex, which contains host proteins as well as virus proteins, is moved along microtubules towards the nucleus.

 Early proteins : nef, tat,

Pox virus
Group I dsDNA The viruses of the pox group are the largest viruses of all. (230 -400 nm ) Human pathogens :
Orthopox: (Vaccinia, Monkey pox and small pox) Parapox: Yatapox: Molluscipox:

Virion structure
Complex structure
Extracellular enveloped virion (EEV) intracellular mature vaccinia virion (IMV)

Envelope (300--thick)
Some argue that there are two closely opposed lipid bilayer membranes; others argue that there is one. extracellular enveloped virion (EEV), contains an additional lipid bilayer membrane that is wrapped around the entire IMV particle

Inner core, dumbbell-shaped. The core contains the double-stranded viral DNA genome and virion enzymes (DNA-dependent RNA polymerase and RNA-processing enzymes) The genome (191-kbp) Ends are covalently connected by single-stranded AT-rich hairpin loops of 101 nucleotides

Replication
It is the only DNA viruses that replicate in a defined area within the host-cell cytoplasm, a so-called virus factory Entry : receptors for the poxvirus are thought to be Glycosaminoglycans (GAGs). Primary un coating

 On release into the host-cell cytoplasm, the core synthesizes viral early mRNAs which are translated by the cellular protein-synthesizing machinery Secondary un coating Coat disassembly Early proteins produced, catalys the viral genome replication and expression of intermediate genes Intermediate gene products controls late genes Late genes produce viral capsid proteins and other enzymes The initial assembly reactions result in formation of the immature virion which is a spherical particle delimited by a membrane that may be acquired from an early compartment of the cellular secretory pathway. This virus particle matures into the brickshaped IMV The IMV can be released by cell lysis Or IMVs acquire a second, double membrane from a trans-Golgi or early endosomal compartment to form the intracellular enveloped virion (IEV) IEVs move to the cell surface on microtubules where fusion with the plasma membrane forms cell-associated virions (CEV). These CEV induce an actin polymerization that promotes a direct transfer to surrounding cells or they can also dissociate from the membrane as EEV.

Replication of bacteriophages
RNA phages
Page MS2 Icosahedral, 25nm, with 180 copies of coat protein Infects E. coli Genome Ss+RNA WITH 3500 nucleotide long, encoding only 4 proteins Maturation proteins, coat proteins, lysis proteins and RNA replicase subunit

 The T-even phages of E. coli use cell wall lipopolysaccharides or proteins as receptors more tail fibers make contact, the baseplate settles down on the surface After the baseplate is seated firmly on the cell surface, conformational changes occur in the baseplate and sheath, and the tail sheath reorganizes so that it shortens from a cylinder 24 rings long to one of 12 rings. the sheath becomes shorter and wider, and the central tube or core is pushed through the bacterial wall. Finally, the DNA is extruded from the head, through the tail tube, and into the host cell. The tube may interact with the plasma membrane to form a pore through which DNA passes.

 Soon after phage DNA injection E.coli RNA polymerase (see section 12.1) starts synthesizing phage early mRNA within 2 minutes. Some early virus specific enzymes degrade host DNA to nucleotides providing raw material for virus DNA synthesis.
Hydroxymethylcytosine (HMC)

Within 5 minutes, virus DNA synthesis commences. Virus gene expression follows an orderly sequence Within 10-12 min. RNA polymerase bind to late promoters and transcribe late genes During the synthesis of DNA several copies of DNA binds together in linear fashion forming a concatamer During assembly, concatemers are cleaved in such a way that the genome is slightly longer than the T4 gene set The sequence of genes in each T4 virus of a population is the same but starts with a different gene at the 5' end.

 The Assembly of Phage Particles

The assembly of the T4 phage is an exceptionally complex selfassembly process.

Late mRNA directs the synthesis of three kinds of proteins:

(1) phage structural proteins, (2) proteins that help with phage assembly without becoming part of the virion structure, and (3) proteins involved in cell lysis and phage release.

 Release of Phage Particles

Many phages lyse their host cells at the end of the intracellular phase. Several T4 genes are involved in this process.
Holin produces a plasma membrane lesion Endolysin attack the peptidoglycan and forms holes in the membrane