Transcript Segmented, dsRNA, Retroviruses

RNA viruses
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Polarity (+ sense or – sense)
Size of genome
Segmented or not
Site of replication
FAMILIES of NEGATIVE STRAND VIRUSES
NON-SEGMENTED (-)STRAND VIRUSES
RHABDOVIRIDAE Rabies, VSV, & Plant viruses
FILOVIRIDAE - Marburg & Ebola viruses
PARAMYXOVIRIDAE - Measles, Mumps, RSV, & Distemper
BORNAVIRIDAE – Neurological diseases of humans and many animals
SEGMENTED (-)STRAND VIRUSES
ORTHOMYXOVIRIDAE - Influenza virus
SEGMENTED AMBISENSE VIRUSES
BUNYAVIRIDAE - Hantavirus, plant Tospovirus and Tenuivirus
ARENAVIRIDAE - Lassa fever
Objectives
• Segmented negative sense RNA viruses:
– Orthomyxoviruses (Influenza virus A, B and C)
– Bunyaviruses (include Hantavirus genus)
– Arenaviruses
• Double stranded RNA viruses
– Reovirus (Rotavirus)
• Retroviruses
– HIV
SEGMENTED NEGATIVE STRAND
VIRUSES
What does segmented mean?
• Genome in segments often representing
different genes.
• Segmented genomes confer evolutionary
advantages.
• Different strains of a virus with a segmented
genome can shuffle and combine genes and
produce progeny viruses or (offspring) that
have unique characteristics.
• This is called reassortment
Viruses with ambisense genomes
• The RNA segments of some of the segmented genome viruses are
ambisense, where one or more of the RNA segments each encodes two
genes, one in the + sense and one in the - sense.
• This is the case for the two genome segments of viruses in the family
Arenaviridae and the family Bunyaviridae, such as tomato spotted wilt
virus; two of the three genome segments of this virus are ambisense.
• The - sense gene of an ambisense RNA is expressed by transcription of a
mRNA.
• The + sense gene is expressed by synthesis of an RNA complementary to
the genome, followed by transcription of the mRNA for that gene.
Segmented Negative Strand RNA Viruses
• Orthomyxoviridae
– Three types of flu virus
• Genus Influenzavirus A – 8 genome segments
• Genus Influenzavirus B - 8 genome segments
• Genus Influenzavirus C - 7 genome segments, no
neuraminidase
– Several insect-transmitted viruses
• Genus Thogotovirus - 6 genome segments
– Thogoto virus – tick-transmitted
– Dhori virus – tick-transmitted
– Batken virus – mosquito-transmitted
Segmented Negative Strand RNA Viruses
• Arenaviridae
– Relatively small group
– Unique characteristic of encapsidating host
ribosomes
– Often associated with persistent infections of rodents
– Several viruses associated with hemorrhagic fevers
– Ambisense genomes
• Bunyaviridae
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Large group of 200+ viruses
Infect vertebrates, invertebrates, plants
Major component of classic “arbovirus group”
Biology usually involves vectors
Many have ambisense genomes
ORTHOMYXOVIRUSES
(ORTHOMYXOVIRIDAE)
• There are three groups of influenza virus: A, B and C.
• Influenza A virus is most intensively studied and influenza A and B are the
most important in human disease.
• Influenza viruses are
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pleomorphic virions (that is, they vary in shape).
negative-sense,
single-stranded RNA
RNA genome that is SEGMENTED, eight RNA segments in influenza A.
nucleocapsid is helical
Enveloped
virions contain RNA polymerase packaged within the virus particle
• Two enveloped membrane glycoproteins (figure 19):
– HA - hemagglutinin - This is the attachment and fusion protein
– NA - neuraminidase - This is important in release, it removes sialic acid from
proteins of the virus and the host cell
Influenza etiology
• Spread person-to-person by aerosol, direct or
indirect contact, in water – no vectors
• Incubation period 1-3 days
• Causes myalgia, sore throat, fever, headache,
cough which may be protracted
• Symptoms typically last 2-7 days
• Intensity of symptoms differs greatly depending
on virus strain
Adsorption and penetration
• The virus adsorbs to receptors on the cell
surface and is internalized by endocytosis.
• At acid pH of an endosome, HA undergoes a
conformational change and fusion occurs.
• Nucleocapsids are released to cytoplasm.
Transcription, translation and
replication
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Nucleocapsids are transported into the
nucleus.
mRNA synthesis and replication of viral
RNA occurs in the nucleus.
This is very unusual for an RNA virus.
Influenza virus has an unusual
mechanism for acquiring a methylated,
capped 5'end to its mRNAs.
A viral endonuclease (which is packaged
in the influenza virus) snips off the 5'end
of a host capped, methylated mRNA
about 13-15 bases from the 5' end and
uses this as a primer for viral mRNA
synthesis.
Hence all flu mRNAs have a short stretch
at the 5' end which is derived from host
mRNA.
Cap Snatching
• The viral RNA polymerase (transcriptase) extends the primer and copies
the template into complementary plus sense mRNA and adds a poly(A)
tail.
• Transcription results in 8 primary transcripts, one transcript per segment.
• Some segments give rise to primary transcripts which can be alternatively
spliced (since influenza virus RNA synthesis occurs in the nucleus, it has
access to splicing machinery), each giving rise to two alternative
transcripts.
• For example, the M segment gives rise to two alternative mRNAs. These
code for the M1 protein and the M2 protein. Thus a single segment can
code for more than one protein since the virus has access to splicing
machinery.
• The mRNAs are translated in the cytoplasm.
• Transmembrane proteins are moved to the plasma membrane while
proteins needed for RNA replication are transported to the nucleus.
Replication of RNA
• RNA replication occurs in the nucleus using a
virus-coded enzyme.
• A full length, exact complementary copy of virion
RNA is made - this + sense RNA is probably
coated with nucleocapsid protein as it is made.
• Full length + strand RNA is then used as a
template for full-length - strand synthesis
• The new - strand is probably coated with
nucleocapsid protein as it is made.
• New - strands can be used as templates for
replication, mRNA synthesis, or packaged.
PROPERTY
PARAMYXOVIRIDAE
ORTHOMYXOVIRIDAE
Genome
non-segmented
segmented
RNA synthesis
cytoplasmic
nuclear
Need for mRNA primer
no
yes
Hemagglutinin,neuramini
dase
if both, part of same
protein (HN)
Influenza A and B have both
but on 2 different proteins
(HA and NA)
Syncytia formation
yes (F functions at at
normal physiol. pH)
no (HA functions at acid pH)
Assembly
• This occurs at the plasma membrane.
• Nucleocapsids are transported out of the nucleus
while envelope proteins are transported via the
Golgi body to the plasma membrane.
• The M1 protein interacts with both nucleocapsid
and a modified region of the plasma membrane
which contains the glycoproteins HA and NA.
• Virus then buds out through the host cell
membrane.
DOUBLE STRANDED RNA VIRUSES
Reoviruses
• Icosahedral symmetry
• Multiple layered capsid
(inner and outer capsid)
• RNA is double stranded.
• There are 10-12 segments
(depending on the genus of
the Reovirus family)
• Due to their clinical
importance in humans, we
shall focus on rotaviruses.
Rotavirus replication
• Rotaviruses infect cells called enterocytes at the ends of the villi
(finger-like extensions) in the small intestine.
• Newly synthesized (+) RNAs enter the cores, and a rigorous
selection procedure ensures that each core receives one each of
the 11 RNA species, a full genome complement.
• Involves the recognition of a unique sequence in each genome
segment.
• Synthesis of (−) RNA takes place during the entry of the (+) strands
into the core, VP1 again acting as the RNA polymerase.
• The dsRNA of the infecting virion therefore remains intact and the
mode of replication is conservative.
• VP6 is added to the core, forming the second layer of the capsid.
• The resulting structure is a double-layered particle similar to that
derived from the infecting virion.
Transcription and translation
• Double stranded RNA does not function as an mRNA - make
mRNA (transcription).
• The mRNAs are made by virally-coded RNA polymerase
packaged in the virion.
• The RNA is capped and methylated by virion packaged
enzymes.
• The mRNAs are translated and the resulting viral proteins
assemble to form an immature capsid.
• The mRNAs are packaged into the immature capsid and are
then copied within the capsid to form double stranded
RNAs.
• More mRNAs are now made by the newly formed
immature capsids.
Assembly
• More proteins are made and eventually the
immature capsids bud into the lumen of the
endoplasmic reticulum.
• They acquire a transient envelope which is lost as
they mature.
• This is a very odd feature of the rotaviruses.
• They are released via cell lysis.
• NOTE: THE ENTIRE REPLICATION CYCLE OCCURS
IN THE CYTOPLASM
Retroviruses
Proteins
capsid
IN = integrase
matrix
NC = nucleocapsid
PR = protease
RH = ribonuclease H
RT = reverse transcriptase
SU = surface glycoprotein
TM = transmembrane glycoprotein
Retrovirus Virion
• Contains two copies of the RNA genome = diploid?
• The two molecules are present as a dimer, formed by base
pairing between complementary sequences .
• The regions of interaction between the two RNA molecules
have been described as a ‘kissingloop complex’.
• As well as the virus RNA, the virion also contains molecules
of host cell RNA that were packaged during assembly. This
host RNA includes a molecule of transfer RNA (tRNA) bound
to each copy of the virus RNA through base pairing.
• The sequence in the virus RNA that binds a tRNA is known
as the primer binding site (PBS)
• Each retrovirus binds a specific tRNA .
Enzyme Activity
• A number of protein species are associated with
the RNA. The most abundant protein is the
nucleocapsid (NC) protein, which coats the RNA,
while other proteins, present in much smaller
amounts, have enzyme activities.
– RNA-dependent DNA polymerase (reverse
transcriptase; RT)
– DNA-dependent DNA polymerase
– Ribonuclease H (RNase H)
– Integrase
– Protease
• Reverse transcription takes place within the reverse
transcription complex
• Synthesis of both the (−) DNA and the (+) DNA begins
at the 3–OH 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.
• The PPT becomes accessible as a result of hydrolysis of
the genome RNA from the 3 end by the RNase H, which
is an enzyme that specifically digests RNA in RNA–DNA
duplexes.
• During synthesis of the two DNA strands, each
detaches from its template and re-attaches at the
other end of the template through base pairing.
• The DNA that results from reverse transcription
(the provirus) is longer than the RNA genome.
• Each of the termini has the sequence U3–R–U5,
known as a long terminal repeat (LTR), one
terminus having acquired a U3 sequence and the
other a U5 sequence.
1. A copy of the virus
genome with a tRNA
bound at the PBS.
2. The reverse transcriptase
begins (−) DNA synthesis
at the 3 end of the tRNA.
3. The RNase H digests the
RNA from the RNA-DNA
duplex. The (−) DNA
attaches at the 3 end of
either the same strand or
the second copy of the
genome.
4. Elongation of the (−) DNA
continues, while the
RNase H degrades the
template RNA from the 3
LTR: long terminal repeat. PBS: primer binding site. PPT: polypurine
tract (a sequence made up entirely, or almost entirely, of purine residues). end as far as the PPT.
R: repeat sequence. U3: unique sequence
at 3 end of genome. U5: unique sequence at 5 end of genome.
5. Synthesis of (+) DNA
begins.
6. The remaining RNA is
degraded.
7. The (+) DNA detaches
from the 5 end of the (−)
DNA template and attaches
at the 3 end.
8. Synthesis of both DNA
strands is completed
Integration of the provirus
• The provirus, still associated with some virion protein, is transported to
the nucleus as a pre-integration complex .
• For most retroviruses this can occur only if the cell goes into mitosis, and it
is likely that mitosis-induced breakdown of the nuclear membranes is
necessary for the pre-integration complex to enter the nucleus.
• This means that there can be a productive infection only in dividing cells.
• HIV and related viruses, however, can productively infect resting cells, as
the pre-integration complexes of these viruses are able to enter intact
nuclei.
• One of the virus proteins still associated with the provirus is the integrase;
this enzyme cuts the DNA of a cell chromosome and seals the provirus
into the gap.
• The integrated provirus genes may be expressed immediately, or there
may be little or no expression of viral genes, in which case a latent
infection has been initiated.
• If a latently infected cell divides, the provirus is copied along with the cell
genome and each of the daughter cells has a copy of the provirus.
Transcription and genome replication
• The two LTRs of the provirus have identical sequences, but
are functionally different; transcription is initiated in one
and terminated in the other.
• Transcription factors bind to a promoter in the upstream
LTR, then the cell RNA polymerase II starts transcription at
the U3–R junction.
• Transcription continues into the downstream LTR.
• There is a polyadenylation signal in the R region and
transcription terminates at the R–U5 junction
• Each transcript is capped and polyadenylated.
• Some transcripts will function as mRNA and a proportion of
these become spliced; others will become the genomes of
progeny virions.
Negative Strand Viruses
• Contain enzymes for transcription in virion
• Make mRNA prior to antigenome
– Message gets capped; genome does not
• Plus strand is template for minus strand
genome
• Makes more minus than plus strand
• 4. Types of Viral Genomes and Their Replication
– Two events critical to viral infection:
• The production of virus structural proteins and enzymes
• Replication of the viral genome (dsDNA, ssDNA, dsRNA, ssRNA)
Figure 3-5
dsDNA Viruses
• Contain dsDNA genome
• Most dsDNA viruses replicate their genomes in the
nucleus of the cell
– Use host’s DNA and RNA synthesizing machinery
Figure 3-6
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
ssDNA Viruses
• Contain ssDNA genomes
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
Figure 3-6b
ss/dsDNA Viruses (Using an RNA
intermediate)
• Virus carries it’s
own reverse
transcriptase
• dsDNA enters the
nucleus, forms an
episome
• Virus does not
encode an
integrase gene
Figure 3-8
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
RNA Viruses
• Genomes may be ss or ds, (+) or (-) sense
• The type of genome determines if the first step after
uncoating will be translation, transcription, or RNA
replication.
• RNA viruses carry an RNA-dependent RNA polymerase that
will synthesize viral genomes into the host cell with them.
Figure 3.9: Differences between positive (+) and negative (-) sense ssRNA viral genomes.
dsRNA viruses
• Contain dsRNA segmented genomes
• Viral polymerase
Figure 3.10: List of dsRNA viruses and their replication strategy.
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
+ssRNA Viruses
• Contain +ssRNA nonsegmented
genomes
• The RNA in the virus particle
functions as mRNA
• Viral mRNA is recognized by
cellular translational machinery
• Contain a viral RNA-dependent
RNA polymerase in order to
replicate viral genomes
Figure 3.11: List of +ssRNA viruses
and their replication strategy.
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
-ssRNA viruses
• Contain -ssRNA segmented or nonsegmented genomes
• Contain a viral RNA-dependent RNA polymerase gene
Figure 3-12b
Figure 3-12a
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
Viruses with ssRNA Genomes That Use
a dsDNA Intermediate to Replicate
• Unique biology
• Viral genome is reverse transcribed and integrated as a cDNA
into the host’s chromosome
Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.
Figure 3-13
3.3 The Error-Prone RNA Polymerase
Genetic Diversity
• RNA viruses mutate or evolve more rapidly than
DNA viruses.
– RNA Polymerases lack proofreading ability
• Most mutations are lethal
– Some mutations are nonlethal
• Selective advantage