Lecture 2 BSCI437. VIRAL GENOMES, PROTEINS, AND LIPIDS.
GENOMES
Criteria for viral genomes
-Same genetic code as host must be used
-Any modifications (polyadenylation of mRNA, capping, splicing) must either depend on
viral enzymes or host enzymes
-There is continuous pressure to minimize size
-fast replication (especially important in bacteria where virus must keep up with host)
-genome packaging (it takes a capsid of several million Daltons to package a 10
kb genome. The larger the genome the larger the capsid must be and this means
more energy and time required for synthesis).
Despite this we see many different kinds of viruses. Some like parvoviruses (ssDNA
about 5000 bases) depend nearly completely on cellular enzymes. They use host cell
DNA polymerases and other host enzymes to replicate. The consequence is they can only
replicate in dividing cells. At the other extreme are Herpesviruses and Poxviruses
(dsDNA 150,000-250,000 bp) that code for over 70 proteins including all the proteins
required to complete DNA synthesis and other specialized proteins that are required for
only some cell types. The gain is that herpes does not depend on the cell for enzymes
and can multiply in most cell types but the expense is that it has a huge genome.
Genome sizes. Range from encoding as little as 2 proteins (eg. Totiviruses, 4kb) to
encoding over 50 (eg. Pox- and Herpesviruses, >100kb).
Structure & Composition: Generally, any and all possible combinations are known.
Composition. Can be RNA, RNA, and/or any combination thereof!
 DNA or RNA
 DNA with short RNA segments
 DNA or RNA with covalently attached protein (e.g. polio)
Topologies. Includes every possible combination of:
 double stranded or single stranded,
 linear or circular,
 contiguous, segmented, or gapped
 polarity: Single stranded (+) strand, (-) strand, or ambisense
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Phenomena related to viral genomes
Properties of viruses that infect animals relative to genome type
Genome
typeA
Variations
(single molecule
or segmented)B
Size Range
(bases or
base pairs)
Viral or host
polymeraseC
fidelity of
Recombina
replicationD -tionE
ReassortmentF
+ssRNA
linear, single
about 700030,000
viral, no
proofread
low
yes
no
-ssRNA
linear, single or
segmented
about 800020,000
viral, no
proofread
low
no
yes, segmented
only
dsRNA
segmented
about 22,000
bp
viral (?)
low?
?
yes
retrovirus
(+ssRNA)
linear
about 10,000
viral, no
proofread
low
yes
no
ssDNA
linear
5000-10,000
host,
proofread
moderate
yes
no
dsDNA
circular or linear
about 35,000300,000
host or viral
high
yes
depending on
particular
virus,
proofread
A
ss-single stranded; ds-double stranded; +/- -plus or minus polarity, plus is same
sequence as mRNA while minus is complement of that sequence; retrovirus- RNA virus
that goes through a DNA intermediate during replication.
B
single molecule indicates a single nucleic acid molecule makes up the whole genome
complement while segmented viruses include 2 or more nucleic acid molecules for a
whole genome complement.
C
Host, polymerase is supplied by infected cell; viral- virus codes for its own polymerase;
proofreading activity implies that the polymerase has a 3'-5' exonuclease activity that can
remove mistakes made during polymerization.
D
Fidelity is in relative terms, low implies error rates of greater than about 1 in 50,000
incorporation events; medium around 1 per 100,000 and high, less than 1 error per
100,000 incorporations
E
recombination by definition is: Exchange of genetic information from two or more virus
genomes to produce recombinant progeny. Recombination of double stranded DNA
generally occurs by cross-over (breakage and reunion) while RNA recombination occurs
via "copy-choice" mechanism during replication.
F
Reassortment by definition is: Exchange of genome segments in segmented viruses.
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no
Special properties of viral genomes.
Terminal Redundancy: genomes of many viruses are terminally redundant. Used as
tools for replication, expression, integration into host chromosomes, and for
protection of ends. Examples include λ, retro-, adeno-, parvo-, pox-, bunya-, and
arenaviruses.
Covalent Modifications: Includes modifications to nucleic acids (eg. methylation,
pseudouridylation, etc.), and covalent linkage with proteins. Of the latter, proteins
covalently linked to the 5' ends of picorna- and adenoviruse RNAs play important
roles in cap-independent translation.
How do viruses condense their genomes?
-Use host proteins for some or all replication functions
-Overlapping genes
-genes on both strands of dsDNA in opposite directions
-multiple splicing of the same transcript to make many different proteins (only need 1
promoter)
-polyprotein production from one mRNA and subsequent proteolytic cleavage
-frameshift mechanisms allow downstream out of frame genes to be made at appropriate
proportions
How do viruses adapt and evolve?
By mutating like other organisms do. Three major phenomena are used:
1. Base misincorporations by polymerases.
2. Recombination by breakage and religation in all DNA viruses or RNA viruses with a
DNA intermediate or by Copy-choice with many ssRNA viruses (see below).
3. Reassortment in the case of viruses with multipartite genomes (more than one
segment)
Note that mutation rate vary greatly between different viruses, as high as about 10-3-10-4
per base for HIV and other ssRNA viruses and as low as 10-8-10-11 for herpesviruses.
The latter are equivalent to what is seen in cellular DNA. The different rates reflect
several things:
-RNA dependent DNA (RT) or RNA polymerase have no 3'-5' proofreading activity so
they don’t correct mistakes.
-RNA viruses cannot access other cellular proof reading enzymes that can correct DNA
errors.
-Polymerases vary in their level of intrinsic accuracy.
Note that there are both benefits and disadvantages to mutations. Escape of the immune
response or drug therapy is a clear advantage while loss of potential progeny, since most
mutations are deleterious is a clear disadvantage.
Virus mutation types
Viruses are subject to the same type of mutations as other organisms (ie. transitions and
transversions, deletions, insertions, nonsense mutations). These mutations can be
spontaneous or induced. Inducing agents like nitrosoguanidine, hydroxylamine, and
nitrous acid are commonly used to directly mutate the virus. Mutations can also be
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induced in vivo by intercalating agents (ethidium bromide for example) or UV irradiation.
Mutations can also be produced by site-directed mutagenesis. Mutations can be used to
map genes in viruses just as they are used to map in bacteria. Recombination of markers
(marker rescue by adding segments also) in viruses with one genome segments and
reassortment in those with multiple. Mutations are also useful in determining the
function of a protein. Most useful in this regard are conditional mutants- a mutant
phenotype that is replication competent under “permissive” but not “restrictive” or
“nonpermissive” conditions. Most common among these are temperature-sensitive
mutants (ts) which can functions at low temperature but not at higher temperature while
wild types can function at both. These are particularly useful because they can occur in
essential genes whose functions are required for replication. We can often spot either ts
or slow growing mutants by observing plaque morphology. Slow growing mutants
produce less progeny so give smaller plaques. Ts mutants stop growing (or slow down if
they are leaky) at the nonpermissive temp. All the mutations are subject to reversion
either at the same or a different (pseudorevertant) location in the genome. Mutants can
also be complemented by other viral strains in a superinfection. True complementation
occurs in the absence of genome alteration (explain with a pol mutant of polio).
Genome related phenomena
Reassortment- Exchange of genome segments in segemented viruses. The most well
known case is influenza which undergoes Antigenic Shift and Drift. Reassortment of
viral genome components (there are 8 segments) can occur when to different viral strains
infect the same cell (Shift). This can lead to the abrupt appearance of new serotypes
which may cause an epidemic. Spontaneous point mutations cause antigenic drift which
is generally less marked. The mixing pot for most flu strains is the swine. It can be
infected by influenza viruses from birds (avian) and humans. Within the infected pig
these viruses can potentially co-infect (superinfect) the same cell and exchange envelope
protein segments. A new virus might have some segments from avian and some from
human virus. In some cases the new virus (or a related variant) can infect humans which
will have no prior immunity to the new virus.
Transduction- Incorporation of cellular genes into the virion genome. It is by this process
that many retroviruses have captured cellular genes. This finding was important in
defining oncogenes. A number of retroviruses carrying viral oncogenes originally
captures from host cells have been discovered. We will discuss latter.
-Attenuation- Process by which a virulent virus losses its ability to cause the disease
normally associated with it but retains the ability to replicate well enough to induce
immunity in the host organism. A number of vaccines have been generated in this way
(poli, measles, rubella). This can occur by serial passage of the virus in an unnatural
host. Examples are passage of poli and rubella viruses in monkey kidney cells and
yellow fever and mumps virus in chick embryo cells. The process of attenuation is not
well understood. Most attenuated viruses have acquired several mutations during passage
so it is difficult to determine which ones contribute to the phenotype.
-Recombination- Exchange of genetic information from two or more virus genomes to
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produce recombinant progeny. Recombination of double stranded DNA generally occurs
by cross-over (breakage and reunion) while RNA recombination occurs via "copychoice" mechanism during replication. (Illustrate a proposed copy-choice mechanism for
poliovirus).
Non-genome related
Viral Interference- The inhibition of replication or infection of one type of virus by
another. Common types are titration of cell surface receptors so that viruses that use the
same receptor cannot enter the cell. Example is HIV which prevents expression of the
CD4 molecule on the cell surface of the infected cell. This may be accomplished directly
by binding of the CD4 molecules at the cell membrane or through the prevention of
export of CD4 which may be mediated by the nef protein of HIV. This prevents
superinfection (infection of a host with more than one virus) of HIV infected cells by
viruses that use the same receptor. This phenomenon has been used to determine if two
viruses with the same tropism use the same receptor to enter the cell. Other viruses
prevent super infection by processes that normally affect the host cell. For example, cells
infected with poliovirus cannot be infected with viruses that have capped mRNAs.
Phenotypic mixing- A phenomenon in which viruses exchange their outside envelopes or
coats. Thus, the genome of one virus may be encapsidated in the external membrane or
coat of the other. In some case the progeny virus is a chimera of the parent viruses while
in other the genome of one virus may be completely encapsidated by the coat or envelope
of another. Although the process can occur between viruses of different genus it most
often occurs within the same genera. This phenomenon can potentially allow viruses that
are tropic for one animal type to be able to now infect another (even humans). An
example is mouse xenotropic virus (MXV) and Avian leukemia virus (ALV) (both
retroviruses. Both viruses can infect duck cells. Superinfection of duck cells with these
viruses gives rise to progeny viruses that can now transfer the avian virus to a wide
variety of mammalian cells and the mouse retrovirus into several different avian cells.
Note that the envelope allows the so called "pseudo-types" to penetrate the different cell
types with appropriate receptors. This does not necessarily mean replication will occur.
Viral Proteins: Viruses can have as few as 2 and as many as >50 virus-encoded proteins.
Generally divided into “Structural” and “Non-structural”.
Structural: These compose the capsids/nucleocapsids, and envelope proteins.
Primary function of those involved in capsid/nucleocapsids is to serve as building
blocks for the virion (viral particle). Envelope proteins are typically glycoproteins
in the form of spikes or projections. Typically, these serve as receptors for host
cell-surface glycoproteins and are involved in viral attachment and entry into cells
(infection).
Non-structural: proteins with enzymatic, virus replicative, or for interactions with
host-cell encoded factors. Examples from HIV include Pol, Int, RNase H,
Integrase, Nef, Vif, and Tat.
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Viral Lipids: viral envelopes contain complex mixtures of neutral lipids, phospholipids
and glycolipies. As a rule, their composition resembles that of the host cell membrane
from which the envelope was derived.
Other Host encoded molecules: Many viruses also contain host-encoded biomolecules.
Examples include tRNAs (used for priming of reverse transcriptase by retroviruses),
5S rRNA and trans-acting factors involved in translation initiation.
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