RNA viruses Polarity (+ sense or – sense) - PowerPoint Presentation

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RNA viruses

Polarity (+ sense or – sense)

Size of genome

Segmented or not

Site of replication

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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

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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

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SEGMENTED NEGATIVE STRAND VIRUSES

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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

 

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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.

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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

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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

Large group of 200+ viruses

Infect vertebrates, invertebrates, plants

Major component of classic “arbovirus group”

Biology usually involves

vectors

Many have ambisense genomes

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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

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

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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

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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.

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Transcription, translation and replication

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.

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Cap Snatching

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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.

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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.

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PROPERTY

PARAMYXOVIRIDAE

ORTHOMYXOVIRIDAE

Genome

non-segmented

segmented

RNA synthesis

cytoplasmic

nuclear

Need for mRNA primer

no

yes

Hemagglutinin,neuraminidase

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)

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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.

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DOUBLE STRANDED RNA VIRUSES

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Reoviruses

Icosahedral symmetryMultiple 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.

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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

.

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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

.

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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

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Retroviruses

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Proteins

capsid

IN

=

integrase

matrix

NC =

nucleocapsid

PR

= protease

RH

=

ribonuclease

H

RT

= reverse

transcriptase

SU

= surface

glycoprotein

TM

=

transmembrane

glycoprotein

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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 .

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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

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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.

Slide35

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.

Slide36

LTR: long terminal repeat. PBS: primer binding site. PPT: polypurinetract (a sequence made up entirely, or almost entirely, of purine residues). R: repeat sequence. U3: unique sequenceat 3 end of genome. U5: unique sequence at 5 end of genome.

A copy of the virus genome with a

tRNA

bound at the PBS.

The reverse transcriptase begins (−) DNA synthesis at the 3 end of the

tRNA

.

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.

Elongation of the (−) DNA continues, while the

RNase

H degrades the template RNA from the 3 end as far as the PPT.

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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

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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.

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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

.

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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

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4. Types of Viral Genomes and Their ReplicationTwo events critical to viral infection:The production of virus structural proteins and enzymesReplication of the viral genome (dsDNA, ssDNA, dsRNA, ssRNA)

Figure 3-5

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dsDNA Viruses

Contain dsDNA genomeMost dsDNA viruses replicate their genomes in the nucleus of the cellUse host’s DNA and RNA synthesizing machinery

Figure 3-6

Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.

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ssDNA Viruses

Contain ssDNA genomes

Figure 3-6b

Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.

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ss/dsDNA Viruses (Using an RNA intermediate)

Virus carries it’s own reverse transcriptasedsDNA enters the nucleus, forms an episomeVirus does not encode an integrase gene

Figure 3-8

Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.

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RNA Viruses

Genomes may be ss or ds, (+) or (-) senseThe 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.

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dsRNA viruses

Contain dsRNA segmented genomesViral polymerase

Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.

Figure 3.10: List of dsRNA viruses and their replication strategy.

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+ssRNA Viruses

Contain +ssRNA nonsegmented genomesThe RNA in the virus particle functions as mRNAViral mRNA is recognized by cellular translational machinery Contain a viral RNA-dependent RNA polymerase in order to replicate viral genomes

Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.

Figure 3.11: List of +ssRNA viruses and their replication strategy.

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-ssRNA viruses

Contain -ssRNA segmented or nonsegmented genomesContain a viral RNA-dependent RNA polymerase gene

Figure 3-12a

Figure 3-12b

Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.

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Viruses with ssRNA Genomes That Use a dsDNA Intermediate to Replicate

Unique biologyViral genome is reverse transcribed and integrated as a cDNA into the host’s chromosome

Figure 3-13

Adapted from D. R. Harper. Molecular Virology, Second Edition. BIOS Scientific Publishers, 1999.

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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