Download After the synthesis of viral nucleic acid and viral proteins

Lec 3
Virology
*Purification of Virus Particles
Pure virus must be available in order for certain types of studies on the
properties and molecular biology of the agent to be carried out. For
purification studies, the starting material is usually large volumes of
tissue culture medium, body fluids, or infected cells. Generally used
many methods for this purposed :
1-The first step frequently involves concentration of the virus particles by
precipitation with ammonium sulfate, ethanol, or polyethylene glycol or
by ultrafiltration .
2-Hemagglutination and elution can be used to concentrate
orthomyxoviruses . virus can then be separated from host materials by
differential centrifugation, density gradient centrifugation, column
chromatography, and electrophoresis.
3-Viruses can also be purified by high-speed centrifugation in density
gradients of cesium chloride, potassium tartrate, potassium citrate,
or sucrose. The gradient material of choice is the one that is least toxic to
the virus. Virus particles migrate to an equilibrium position where the
density of the solution is equal to their buoyant density and form a visible
band.
4-Additional methods for purification are based on the chemical
properties of the viral surface. In column chromatography, virus is bound
to a substance such as diethylaminoethyl or phosphocellulose and then
eluted by changes in pH or salt concentration. Zone electrophoresis
permits the separation of virus particles from contaminants on the basis of
charge.
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5-Icosahedral viruses are easier to purify than enveloped viruses. Because
the latter usually contain variable amounts of envelope per particle, the
viral population is heterogeneous in both size and density.
*Replication of Viruses:
Viruses multiply only in living cells. The host cell must provide the
energy and synthetic machinery and the low-molecular-weight
precursors for the synthesis of viral proteins and nucleic acids. The viral
nucleic acid carries the genetic specificity to code for all the virusspecific macromolecules in a highly organized fashion.
In order for a virus to replicate, viral proteins must be synthesized by the
host cell protein-synthesizing machinery. Therefore, the virus genome
must be able to produce a usable mRNA. Various mechanisms have been
identified which allow viral RNAs to compete successfully with cellular
mRNAs to produce adequate amounts of viral proteins.
After the synthesis of viral nucleic acid and viral proteins, the
components assemble to form new infectious virions. The yield of
infectious virus per cell ranges widely, from modest numbers to more
than 100,000 particles. The duration of the virus replication cycle
also varies widely, from 6 to 8 hours (picornaviruses) to more than 40
hours (some herpesviruses.
Note :Not all infections lead to new progeny virus.
Productive infections occur in permissive cells and result in the
production of infectious virus.
Abortive infections fail to produce infectious progeny, either because the
cell may be nonpermissive and unable to support the expression of all
viral genes or because the infecting virus may be defective, lacking some
functional viral gene.
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A latent infection may ensue, with the persistence of viral genomes, the
expression of no or a few viral genes, and the survival of the infected cell.
The pattern of replication may vary for a given virus, depending on the
type of host cell infected.
*General Steps in Viral Replication Cycles
A variety of different viral strategies have evolved for accomplishing
multiplication in parasitized host cells are :
1- Attachment:
The first step in viral infection is attachment, interaction of a virion
with aspecific receptor site on the surface of a cell. Receptor molecules
differ for different viruses but are generally glycoproteins. In some cases
the virus binds protein sequences (eg, picornaviruses) and in others
oligosaccharides (eg, orthomyxoviruses and paramyxoviruses). Receptor
binding is believed to reflect configurational homologies between a
virion surface structure and a cell surface component.
For example, human immunodeficiency virus(HIV) binds to the CD4
receptor on cells of the immune system,. The presence or absence of
receptors plays an important determining role in cell tropismand viral
pathogenesis. Not all cells in a susceptible host will express the
necessary receptors; for example, poliovirus is able to attach only to cells
in the central nervous system and intestinal tract of high mammals . Each
susceptible cell may contain up to 100,000 receptor sites for a given
virus. The attachment step may initiate irreversible structural changes in
the virion.
2-Penetration:
After binding, the virus particle is taken up inside the cell. This step is
referred to as penetration or engulfment. In some systems, this is
accomplished by receptor-mediated endocytosis, with uptake of the
ingested virus particles within endosomes. There are also examples of
direct penetration of virus particles across the plasma membrane.
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3-Uncoating:
Uncoating occurs concomitantly with or shortly after penetration.
Uncoating is the physical separation of the viral nucleic acid from the
outer structural components of the virion so that it can function. The
genome may be released as free nucleic acid (picornaviruses) or as a
nucleocapsid (reoviruses). The nucleocapsids usually contain
polymerases. Uncoating may require acidic pH in the endosome.
4- Replication :
After uncoating the nuclic acid of virus start replication in
nucleus ( DNA viruses ) or in cytoplasm of host cells by
enzymes of viruses and host cells .
Note : poxviridae replicate in cytoplasms .
5-Assembly& Releasing :
Newly synthesized viral genomes and capsid polypeptides assemble
together to form progeny viruses. Icosahedral capsids can condense in the
absence of nucleic acid, whereas nucleocapsids of viruses with helical
symmetry cannot form without viral RNA. In general, nonenveloped
viruses accumulate in infected cells, and the cells eventually lyse and
release the virus particles.Enveloped viruses mature by a budding
process.
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Figure (1)Receptor-mediated endocytosis ofvirus particle .
Figure (2) Fusion of viral envelope withmembrane of host cell
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*Families of viruses :
***-DNA
Viruses families are :
1- Single strand DNA viruses or ssDNA(e.g : parvovirus or B19
cause fifth disease or Erthema disease )
2- Double strand DNA viruses or dsDNA include :
A-Herpesviridae( e.g : Herpes simplex virus type 1 cause lesion in
fase and lips ,type 2 cause genitel infection ,type 6and 7 are causative
agents of roseola infantum , herpes simplex type 8, cytomegalovirus
cause abortion in women ,Varicella-Zoster virus cause chickenpox in
human and Epstein –Barr virus cause infection in B-lymphocyte )
B-Poxviridae ( e.g :Smallpox in human, Vaccinia virus , Molluscum
contagisum virus )
C-Adenoviridae ( e.g : Adenovirus cause ocular , respiratory
,gastrointestinal and urinary tract infection )
D -Papillomaviridae ( Papilloma virus cause wart in human )
3- dsDNA intermediate RNA viruses called Retroviruses
(Hepadnaviridae e.g :Hepatitis B virus in human ).
Questions expected about this lecture :
1- Explain steps of replication of viruses .
2- Classify the DNA viruses with examples .
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