Danielle Blondel – Laboratoire de Virologie Moléculaire et Structurale UMR CNRS 2472-INRA 1157CNRS- Gif sur Yvette

RNA viruses

I.Introduction to viruses 1. Definition 2. Diversity : nature and structure 3. Classification

II. Animal RNA viruses replication strategies 1. Plus-stranded RNA viruses (Ex : Poliovirus) 2. Negative-stranded RNA viruses 2a) Non –segmented (Ex : Rabies virus) 2b) Segmented (Ex : Influenza virus) 3. Double-stranded RNA viruses (Ex : Rotavirus) 4. Retroviruses 5. Variability of viral genomes

What are viruses?

The viruses responsible of diseasesIn humans and animals

Viral diseases are known since several thousands of years

• 3700 BC : The first written record of a virus infection is from ancient Egypt which shows a temple priest with typical signs of paralytic poliomyelitis.

• 2500 BC : chineses recognized the nature and the specific characters of small pox

• 460 BC : Mumps was described by Hippocrate

• 50 BC : Rabies was described by Virgile and Homère

1796 : Jenner and the vaccination against smallpoxEdward Jenner used cowpox to successfully vaccinate a child.Vaccination against smallpox was almost universally adopted worldwide during the nineteenth century.

1885 : Pasteur and rabies vaccination 1881: Louis Pasteur began to study rabies in animals. Over several years, he developed methods to produce attenuated virus preparations which would protect from challenge with virulent virus.1885, he inoculated a child, with the first artificially produced virus vaccine.

The first success

In plants• Tobacco mosaic virus (VMT) (Ivanowsky 1892/

beijerink 1898)• Tomato mosaic virus

In bacteria• Bacteriophages (1917, 1947, 1952)

Viruses responsible of diseases

Before the definition of virus

1. The agent must be present in every case of the

disease.

2. The agent must be isolated from the host and

grown in vitro.

3. The disease must be reproduced when a pure

culture of the agent is inoculated into a healthy

susceptible host.

4. The same agent must be recovered once again from

the experimentally infected host.

Koch’s Postulates :

Toward the definition of virus

1891 : Ivanowski showed that extracts from diseased tobacco plants could transmit disease to other plants after passage through ceramic filters fine enough to retain the smallest known bacteria. Tobacco mosaic virus (VMT) is filterable

1898 : Beijerinick confirmed and extended Iwanowski's results on Tobacco mosaic virus and was the first to develop the modern idea of the virus as “contagium vivum fluidum” (soluble living germ).

1930 : Electron microscopy determination of the composition, structure and morphology of viruses .

Ex: VMT with rod shaped

Virus definition:

• Virus particles (virions) themselves do not grow or

undergo division

• Viruses have only one type of nucleic acid

They lack the genetic information which encodes

apparatus necessary for the generation of metabolic

energy or for protein synthesis (ribosomes)

They are therefore absolutely dependent on the host

cell for this function.

Therefore, they are absolute parasites

Virus diversity

• There is more biological diversity within viruses than in all the rest of the bacterial, plant and animal kingdoms put together.

• This results comes from the success of viruses in parasitizing all known groups of living organisms.

Classification was required

The basis of the classification

• Initially, classification was based : - on common pathogenic properties - on common organ tropisms and common ecological

and transmission characteristics

• Since 1930, informations of the structure and composition of viruses started to emerge.

Therefore, taxonomy was based on the structure and composition of the virus particle

Now:

Viruses are classified according to the nature andstructure of their genome :

Order : -virales

Family : -viridae

Sub-family : -virinae

Genus : -virus

Ordre Famille Sous famille Genre Hôte / exemple

Mononegavirales

Paramyxovirinae

Paramyxovirus Vertebrate / parainfluenza 1 Virus

Morbillivirus Vertebrate / meales virus

    Rubulavirus Vertebrate / mumps virus

    Pneumovirinae Pneumovirus Vertebrate / respiratory syncitial virus

 

Rhabdoviridae

  Lyssavirus Vertebrate / rabies virus

    Vesiculovirus Vertebrate / vesicular stomaitis Virus

  Ephemerovirus Vertebrate / bovin ephemerale fever virus

Cytorhabdovirus Plants / yellow mosaic virus

  Nucleorhabdovirus Plants / potato yellow dwarf virus

  Filoviridae   Filovirus Vertebrate / Ebola virus

Paramyxoviridae

Mononegavirales order

ICTV Virosphere

Today , there are 4000 viruses

RNA viruses

Lwoff, Horne et Tournier,1962

DNA Viruses

Structures of Viruses

1. Core nucleic acid

2. Protein coat or capsid - composed of a large number of sub-units - role of protection 3. Envelope or not (some are nude)

virion size range is ~10-400 nm in diameter

Viruses contain :

Diversity of viral genomes

1. the nature of the nucleic acid : - DNA

- RNA2. the structure - single-stranded - double-stranded 3. the shape - linear, circular - segmented, non-segmented

1. helical :- rod shaped (structures of plant viruses)- Bacteriophages- Nucleocapsids of enveloped viruses

2. icosahedral - « spherical» viruses

The capsids : two types of symmetryProtective coat made of repeating sub-units of proteins

Two types of viruses /membrane

have a membrane derived from the host cell membrane but modified by insertion of viral proteins (glycoproteins)

• Nude viruses

• Enveloped viruses

Vesicular Stomatitis virus Ebola virusInfluenza virus

Viral cycle

1.Adsorption and attachment to cell membrane via receptors

2. Penetration and uncoating

3. Biosynthesis of viral RNA and proteins

4. Maturation : nucleic acid and viral proteins assembly

5. Release of virions by budding

1. Adsorption and attachment to cell membrane via specific receptors

2. Penetration and uncoating Envelope and capsid are removed and viral nucleic

acid is released into the cell

Viral cycle

The nature of the receptor is responsable of the viral tropismThe receptor can be a protein, a sugar or a complex lipid…

• Enveloped viruses : two possible mechanisms of fusion

Stratégies de décapsidation p 137

Fusion between the viral membrane and the cellular membrane

Fusion between the viral membrane and the endosomal membrane

Non-enveloped viruses may cross the plasma membrane directly or may be taken up into endosomes. They then cross (or destroy) the endosomal membrane.

3. Expression and replication of viral genomes

4. Assembly of viral nucleic acid and viral proteins

5. Release Budding of enveloped virus

RNA viruses replication strategies

The strategy of viral replication depends on the type of nucleic acid involved

Replication of RNA viruses are quite complex

Single-stranded RNA viruses contain either positive-sense or negative-sense RNA.

By convention, positive sense RNA serves as messenger RNA

If positive sense, viral RNA serves as mRNA for production of viral proteins

If negative-sense, no viral proteins can be made until viral mRNA is available then need to make a positive strand (by a viral RNA enzyme)

Then, the different strategies of replication of RNA viruses result in the synthesis of viral messenger RNA

Messenger RNA

RNA ss positivePolarity

PicornaFlaviCaliciAstro

TogaCoronaArteriNodaTetra

Genomic RNA= mARN

Retro

RhabdovirusFiloBornaParamyxoOrthomyxoBunyaViral RdRp

Transcriptaseinverse

dsDNA

RNA pol IIcellulaire

ds RNA

ReoBirna

ambisens RNA

Arena(bunya)

RNA ss negative Polarity

Viral RdRp

Viral RdRp

Positive strand RNA viruses

The genomic RNA is translated by the cellular machinery for translation of viral proteins

In these viruses, the genomic RNA is the same sense as mRNA and so functions as mRNA. This mRNA is translated immediately upon infection of the host cell

examples :

Prototype member : Poliovirus (Picornaviridae)

Human rhinovirus (Picornaviridae)

Hepatitis C (Flaviviridae)

Synthesis of one and large polyprotein precursor of the structural and non

structural by proteolytic clivage

The poliovirus

The genomic RNA has two characteristics :1) The 3’end is polyadenylated 2) The 5’end is not capped but is covalently associated to

a small protein of 22aa (VPg) and it forms a secondary structure called IRES.

The poliovirus has an RNA-dependant RNA polymerase.

AUG nt743VPg

AAAAA

Poliovirus (Picornavirus)

ProteasesRNA synthesis

New virions

Viral proteinsnon structural

Polyprotein precursor of viral proteins

TranslationMaturation cleavage

and structural

Picorna and Flaviviruses

genome(+)

antigenome(-)replication Enzymes

genome(+)

c

replication

5’3’

5’

5’c

+sense

-sense

+sense

Negative strand RNA viruses

The genomic RNA is negative sense (complementary to mRNA) and must therefore be copied into the complementary plus-sense mRNA before proteins can be made.

Thus, besides needing to code for an RNA-dependent RNA-polymerase (RdRp), these viruses also need to package it in the virion so that they can make mRNAs upon infecting the cell.

Non segmented negative strand viruses : the mononegavirales

The genome is a single-stranded RNA of negative polarity (10 à 15kb) associated to the nucleoprotein N.

The first step is the transcription of the genome by the RNA polymerase packaged by the virus and the polymerase that has polyadenylation and capping.

3’ 5’

3’ 5’1. transcription

5’ 3’Antigenome (+)

Genome

2. Replication

Genome(-)

mRNA

Mononegavirales order

Ordre Famille Sous famille Genre Hôte / exemple

Mononegavirales

Paramyxovirinae

Paramyxovirus Vertebrate / parainfluenza 1 Virus

Morbillivirus Vertebrates / meales virus

    Rubulavirus Vertebrates / mumps virus

    Pneumovirinae Pneumovirus Vertebrates / respiratory syncitial virus

 

Rhabdoviridae

  Lyssavirus Vertebrates/ rabies virus

    Vesiculovirus Vertebrates / vesicular stomaitis Virus

  Ephemerovirus Vertebrates / bovin ephemerale fever virus

Cytorhabdovirus Plants / yellow mosaic virus

  Nucleorhabdovirus Plants / potato yellow dwarf virus

  Filoviridae   Filovirus Vertebrates / Ebola virus

Paramyxoviridae

55 000 persons die of rabies each year.

Enveloped virus genome : negative single-stranded

RNA(~12kb)

5 proteins G : glycoprotein

N : nucleoprotein L : RNA polymeraseP : phosphoprotein

M : matrix Nucleocapsid (RNA-N) Helical symetry

Rabies virus

Besides the order of mononegavirales, there are viruses (like orthomyxoviridae) that have many fragments of negative polarity. Their genome are segmented

Example: Influenza virus

Their replication strategies are identical

Segmented negative strand RNA viruses

Influenza virus

Enveloped virusgenome: negative ss segmented RNA (8 segments)

2 glycoproteins:-HA (H1 à H15)-NA (N1 à N9)

HA

NA

M2

M1

M1

NS2

genome

M2

PAPB2 PB1

5’ UUU 3’ viral RNA

3’PAPB2 PB1

cellular mRNA

3’

PAPB2 PB1

Cap-snatching

•The RdRp (PA, PB1, PB2)

has no the activity required

for the capping of the mRNA.

• but the RdRp has endonuclease activity

required to snatch capped primers from

host pre-mRNAs for viral transcription

(Cap-snatching).

Transcription

RNA -dependent RNA Polymerase

packaged in the virion

mARNTranslation

Transcription

mRNA

rhabdo, filo, borna, paramyxo, orthomyxovirus

genome(-) (nucleocapsids)

Progeny virus

replication RNA-dependent RNA polymerase

antigenome(+) (nucléocapsids)

genome(-) (nucléocapsids)

Viral proteins

2 steps:1. Transcription of a part of the genome in messenger RNA encoding capsid proteins and RNA polymerase

2. Transcription of a part of the antigenome in messenger RNA encoding other viral proteins

Examples: arenavirusand some bunyavirus

5’

5’

3’

transcription

5’

mRNA

3’ genome

antigenomemRNA

transcription

There are viruses which contain ambisens genome

Double-stranded RNA Viruses

Example : reoviridae (reovirus and rotavirus)

• Genome with 10 and 11 segments of ds-RNA

• The virus contains a ds RNA dependent RNA polymerase

• The transcription is asymetric : one of the ds-strand is transcribed

• The transcription takes place inside the subviral particle that contains all the activities required for the capping of messenger RNA.

Rotavirus

Nude virustriple capsid

11 segments of dsRNA

6 structural proteins6 non structural proteins

Rotaviruses are found worldwide, causing major diarrhea-associated hospitalization and 600,000-850,000 deaths per year.

RNAdb

ARN(-)

Proteins

Transcription RNA-dependent RNA pol

Packaged in the virion

mRNA

Progeny virus

Reoviridae and Birnaviridae

Partial assembly

double strand RNAprogeny

Retroviridae

These viruses contain a genomic RNA that will be used as a template for the synthesis of DNA by a reverse transcriptase

2 examples:Virus de l’immunodéficience humaine (HIV 1 and 2)Virus de la leucémie des cellules T humaines (HTLV )

Retrovirus

Enveloped virus Genome : RNA

Proteins : products ofGag, Pol, Env genes

mRNA

Proteins

Progeny virus

dsDNAReplication

Retrovirus

Parental RNA

RNA

TranscriptionRNA pol II integrated DNA

(provirus)

Integration in thecellular DNA

linear DNA

tRNA and reverse transcriptase

packaged in the virion

4) 3’5’ 3’

PB’ gag’ pol’ env’ U’3 R’ U’5

3)3’

5’

env’ U’3 R’ U’5

PB gag pol PP

U3 R U5 PB5’

5) 3’ PB’ gag’ pol’ env’ U’3 R’ U’5

6)

U3 R U5 PB5’

U’3 R’ U’5 PB’

7)

LTRLTR

U3 R U5 PB gag pol env U3 R U5

2) 5’3’

3’

R’ U’5

PB gag pol env U3 R

R’ U’5

1) 5’3’

R U5 PB gag pol env U3 R3’

Viral quasispeciesThe virale polymerases (RdRp, RT) are very inaccurate : lack of proofreading mechanism

Error rate : 1/10000

This results in viral variants or quasispeciesand confers significant adaptation potential through the selection of mutants best suited to a new environment.

- Escape from immune responses- Faster replicating, more aggressive strains- Broader cell tropism - Escape to antiviral therapies.