Viruses

1
Viruses
Obligate cellular parasites-- can't replicate
without cells nucleic acid core (DNA or
RNA) surrounded by a protein coat take over
cellular machinery to reproduce themselves and
eventually kill the host cell
2
Viruses
Many viruses cause disease
HIV virus
Ebola virus
Smallpox virus
3
Viruses
Tobacco mosaic virus infects plants excellent
model for studying life cycle viruses can be
used for genetic engineering of plants and animals
bacteriophage are viruses that infect
bacteria vital to early molecular biology
4
What do Viruses have to do to survive?
1) Must recognize a binding partner on the
surface of the cell ie. HIV uses a protein
(gp41) which binds CD4 and CCR5
bacteriophage lambda recognizes the maltose
receptor 2) Must fuse or enter the cell in some
fashion some viruses are covered by a membrane--
direct fusion endocytosis of the virus,
release of viral particles due to pH other
viruses are protein coats-- inject DNA or RNA
only bacteriophage lambda protein is on the
surface, DNA injected 35S labels proteins
(specifically amino acid methionine) 32P labels
DNA (specifically phosphate backbone) in 1952,
Hershey and Chase labeled bacteriophage and
infected bacteria for a short period of time,
then stripped the phage coat by blending 32P was
found in infectious phage particles, very little
35S found there
5
What do Viruses have to do to survive?
3) Viral RNA processing Both DNA and RNA viruses
have to make RNA to replicate viral genomes are
small and compact-- most viruses make
polygenic RNA transcripts polygenic transcripts
are mRNAs that code for multiple proteins like
bacterial operons do IRES site internal
ribosome entry site- specific sequence of mRNA
that allows ribosomes to assemble in the
middle of a mRNA molecule viruses use cellular
RNA polymerases to make their molecules reverse
transcriptase viral enzyme that converts RNA
into DNA required during the life cycle of
RNA viruses chemicals which block IRES sites or
reverse transcriptases are good candidates
for anti-viral therapies
6
What do Viruses have to do to survive?
4) viral proteins must be processed before they
are functional many cellular proteins get
processed as well (ie. methionine is
cleaved, proteins are glycosylated, etc) many
viral proteins are made as part of a large
protein that is cleaved into smaller
functional units some human proteins are also
produced so that they are inactive until
processed ie. zymogens like plasminogen or
trypsinogen viral proteins are processed by
specific proteases (enzymes that cleave
proteins) inhibitors of the viral proteases can
also block replication some viruses have
additional proteins that help them replicate
directly or indirectly influenza has an
enzyme (neuraminidase) which breaks down
specific cell surface carbohydrates enzyme
inhibited by Tamiflu
7
Introduction to RNA interference (RNAi)
RNA interference (RNAi) targeted degradation of
particular mRNAs using a 22-23 bp piece of
double stranded RNA which is complimentary to
the sequence of the mRNA if the double stranded
RNA has some mismatches with the mRNA, it can
simply block translation of that mRNA believed to
have evolved as an anti-viral response to protect
against RNA viruses interferon response
cellular response to 'long' 50bp fragments of
double stranded RNA (found in some viruses)
activates a particular protein kinase PKR that is
usually inactive mRNA naturally occurring RNAs
which can inhibit the translation of different
normal mRNAs mRNAs are regularly found within
introns, so that the transcription of one gene
results in the negative translational regulation
of other genes
8
(No Transcript)
9
RISC digests cognate mRNAs
10
RISC digests cognate mRNAs
11
RISC digests cognate mRNAs
12
RISC digests cognate mRNAs
13
RISC digests cognate mRNAs
14
Methods for Studying RNA
1) convert it into cDNA using reverse
transcriptase easier to handle, more stable,
more versatile, VERY sensitive less
quantitative, indirect 2) in situ
hybridization-- sticking complimentary labeled
DNA probes onto tissue sections or small
tissue blocks to bind to mRNA direct
visualization, gives tissue/cell specificity low
efficiency-- hard to detect, needs a lot of mRNA,
no size info 3) northern blot-- sticking
complimentary labeled DNA probes onto
nitrocellulose blots from mRNA separated by size
on a gel relatively sensitive, process many
samples at once gives size
information difficult to run well, no spatial
separation (all RNA in a tissue)
15
Terminology for reading Ge et al.
titre how much virus is present in a given
sample (often pfu/mL) pfu/mL plaque forming
units/ mL- plaque 1 infectious virus
particle moi multiplicity of infection- of
virus particles per cell HA hemagglutinin
assay- agglutination sticking together NA
neuraminidase- cleaves carbohydrates on cell
surface glycoproteins both of these enzymes are
on the surfaces of the virus particle 15 HA and 9
NA subtypes-- used for naming viruses (ie.
H5N1) lipofection lipid technique used for
transfering DNA or RNA into a cell
alternative to electroporation, chemical
transfection, or transduction oligofectamine
commercial lipid preparation for
lipofection vRNA '-' strand RNA-- is packaged
into the virus makes mRNA '' strand using a
viral RNA dependent RNA polymerase also makes
cRNA (another '' strand) to make more
vRNA influenza has no reverse transcriptase-
doesn't make DNA to integrate