Dr. Margaret Hunt 

MEDICAL MICROBIOLOGY, MBIM 650/720

Reading:  Murray et al., Microbiology, 3rd Ed.: Ch. 6 and appropriate parts of Ch. 54 (Picornaviruses), Ch. 58 (Rhabdoviruses), Ch. 55 (Paramyxoviruses), Ch. 56 (Orthomxyoviruses), Ch. 57 (Reoviruses), Ch. 48 (Vial diagnosis)

  VIROLOGY - LECTURE 4  

  RNA VIRUS REPLICATION STRATEGIES  

TEACHING OBJECTIVES

Descriptive analysis of the replicative strategies employed by animal RNA viruses

Identification of virus prototypes associated with different RNA virus replication schemes

Structure of Polio Type 1 Mahoney. X-ray data from Hogle et al.(Harvard Univ.), PDB entry 2PLV, rendered with GRASP (A.Nicholls, Columbia Univ.). Courtesy of Dr Sgro and the Institute for Molecular Virology, Univ. of Wisconsin  (used with permission)

a) RNA VIRUSES WHICH DO NOT COPY THEIR RNA INTO DNA:

These viruses need an RNA-dependent RNA-polymerase to replicate their RNA, but animal cells do not seem to possess such an enzyme. Therefore, this type of animal RNA virus needs to code for an RNA-dependent RNA polymerase.

No viral proteins can be made until viral messenger RNA is available. Thus, the nature of the RNA in the virion affects the strategy of the virus.

i. plus-stranded RNA viruses

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

Examples:  
poliovirus (picornavirus) 
togaviruses
flaviviruses

ii. negative-stranded RNA viruses

The virion 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, these viruses also need to package it in the virion so that they can make mRNAs upon infecting the cell.

Examples:  
influenza virus (orthomyxovirus)
measles virus, mumps virus, (paramyxoviruses)
rabies virus (rhabdovirus)

iii. double-stranded RNA viruses

The virion (genomic) RNA is double stranded and so cannot function as mRNA; thus these viruses these also need to package an RNA polymerase to make their mRNA after infection of the host cell.

Example:
rotaviruses (belong to reovirus family)

b) RNA VIRUSES WHICH COPY THEIR RNA INTO DNA:

These are the retroviruses. In this case, their virion RNA, although plus-sense, is not released in the cytoplasm, and so does not function as mRNA immediately upon infection. Instead it serves as a template for reverse transcriptase and is immediately copied into DNA. Reverse transcriptase is not available in the cell, and so these viruses need to code for this enzyme and package it in virions.

THE TRANSLATION PROBLEM

Eucaryotic host cell translation protein synthesis machinery in general uses monocistronic mRNAs and so there is a problem in making more than one type of protein from a single mRNA.

RNA viruses have several solutions to this problem:

i. The virus makes multiple monocistronic mRNAs

ii. The virus makes primary transcripts which are processed by the host splicing machinery to give more than one monocistronic RNA

iii. The viral mRNA acts as a monocistronic transcript, a large polypeptide (called a polyprotein) is made which is then cleaved into separate proteins - Thus, one initial translation product is processed to give rise to multiple proteins. This happens, for example, in picornaviruses

 

GENOME SIZE OF RNA VIRUSES

RNA viruses tend to have a relatively small genome (although virion size may not necessarily be small). This is probably because the lack of RNA error correction mechanisms puts a limit on the size of RNA genomes.

The result of having a small genome is that RNA viruses tend to code for only a few proteins. These will include a polymerase which can copy RNA into a complementary nucleic acid (either RNA or, as in the case of retroviruses, DNA) and a viral attachment protein.

Polio virus x 350,000 © Dr Dennis Kunkel, University of Hawaii. Used with permission

Examples: 

picornaviruses
togaviruses
flaviviruses

  1. PICORNAVIRUSES

PROPERTIES

Small (28nm), naked icosahedral viruses (pico=very small)
RNA is single-stranded, plus sense, polyadenylated - functions as mRNA immediately upon infection
Prototype member: poliovirus

ADSORPTION AND PENETRATION

A viral protein recognizes a receptor on the host cell membrane (important in tropism of virus).
It seems that binding to receptor alters capsid structure in some way.
RNA is released into cytoplasm - Details of this process remain unknown.
mRNA is now available for translation.

SYNTHESIS OF VIRAL PROTEINS

Poliovirus virion RNA functions as an mRNA but does not have the methylated cap structure typical of eucaryotic mRNAs - it has a "ribosome landing pad" (internal ribosome entry site, IRES) which enables ribosomes to bind without having to recognize a 5' methylated cap structure.

The mRNA is translated into a single polypeptide (polyprotein), which is cleaved.

Cleavage occurs before translation is complete, i.e. on nascent chain.

Cleavages are carried out by virally coded proteases.

Products of cleavage include:

An RNA polymerase (replicase)
Structural components of the virion
Proteases

RNA REPLICATION

We now have newly made viral proteins to support replication.

1. Viral RNA polymerase copies plus-sense genomic RNA into complementary minus-sense RNA:

This process needs

VPg (or precursor containing VPg)
RNA polymerase (replicase)
Host proteins

VPg may act as a primer for RNA synthesis, this would explain why it is at the 5' end of all newly synthesized RNA molecules

2. New minus sense strands serve as template for new plus sense strands. Again  polio RNA replicase and VPg are needed. VPg is linked to the 5' ends of the new plus sense strands (again, it probably functions as a primer).

New plus strand has three alternative fates:
i. It may serve as a template for more minus strands
ii. It may be packaged into progeny virions
iii. It may be translated into polyprotein (In this case VPg is removed prior to translation)

ASSEMBLY

When sufficient plus-sense progeny RNA and virion proteins have accumulated, assembly begins.
Particles assemble with VPg-RNA inside and 3 proteins in the capsid [VP0,1 and 3].
VP0 is then cleaved to VP2 and VP4 and these mature virions are infectious.
Virions are released following cell lysis

Examples: 

Rhabdoviruses
Paramyxoviruses

Example: Rabies virus. The most intensively studied member is vesicular stomatitis virus.

RNA: 
single stranded
negative (minus) sense
codes for 5 proteins

ATTACHMENT, PENETRATION AND UNCOATING
The virus adsorbs to cell surface.
G (Glycoprotein) is attachment protein which  binds to receptor on host cell.
The attached virus is endocytosed via clathrin coated pits.
The membrane of virus fuses with the endosome membrane (acid pH of endosome is important because an acid pH is required for fusion to occur).
The nucleocapsid is released into cytoplasm.

TRANSCRIPTION:
'Transcription' is used in this context to refer to synthesis of mRNAs.
Complete uncoating of the nucleocapsid is not necessary - the virion RNA polymerase can copy virion RNA when it is in the nucleocapsid form. This is an advantage in that genomic RNA is therefore somewhat protected from ribonucleases.
There is one monocistronic mRNA for each of the five virally coded proteins.
The mRNAs are capped, methylated, and polyadenylated.
Since this is a cytoplasmic, negative-sense RNA virus, the enzymes for mRNA synthesis and modification are packaged in the virion.

TRANSLATION

mRNAs are translated on host ribosomes.
All 5 proteins made.
There is no distinction between early and late functions.

RNA REPLICATION

RNA replication is the process by which new copies of genome-length RNAs are made.
RNA replication occurs in the cytoplasm and is carried out by the viral RNA polymerase.
The full length plus strand is coated with nucleocapsid protein as it is made (mRNAs are not coated with this protein, which would interfere with the host protein translation machinery).

  The new positive strand is copied into full length minus strand, which is also coated with nucleocapsid protein as it is made.
(Note: since the viral RNA polymerase synthesises  mRNAs (transcription) and full-length RNA (replication), it is also sometimes called a transcriptase or a replicase , such names just focus on the different aspects of the polymerase activity.)

New negative strands may:

i. be used as templates for the synthesis of more full length plus strands
ii. be used as templates for the synthesis of more mRNAs
iii. be packaged into virions

ASSEMBLY

The virus consists of two "modules" - the envelope and the nucleocapsid:

Envelope
Transmembrane proteins are made on ribosomes bound to the endoplasmic reticulum. They are inserted into the endoplasmic reticulum membrane as they are made. The transmembrane proteins are glycosylated in the endoplasmic reticulum and pass through the Golgi body where substantial modification of the carbohydrate chains occurs. They are then transported, in vesicles, to the appropriate cell membrane; in the case of vesicular stomatitis virus, this is the plasma membrane.

Nucleocapsid

Synthesis of the nucleocapsid was described above. Once nucleocapsids are formed, the RNA polymerase associates with them - and the complex is now ready to be packaged.

Nucleocapsids bud out through modified areas of membrane which contain G and M proteins. The M (matrix) protein is involved in assembly - it interacts with patches of G in the membrane and with nucleocapsids.

Paramyxoviruses:

Pleomorphic
Negative-sense, nonsegmented RNA, helical nucleocapsid
Enveloped
Envelope contains two virally coded glycoproteins: The F protein and the attachment protein

The F protein has fusion activity.

The attachment protein binds to receptors on the host cell, this protein may have:

hemagglutinating activity and neuraminidase activity (HN protein)
or hemagglutinating activity alone (H protein)
or neither (G protein).

Hemagglutination is easy to test for in the clinical laboratory and is used in diagnosis 

Hemagglutination involves the agglutination of red blood cells. It relies on the ability of a virus to bind to receptors on red blood cells. Since viruses have multiple attachment proteins per virion, they can bind to more than one red blood cell and so they can serve to link red blood cells into a network. Inactivated virus can still hemagglutinate as long as its attachment proteins are intact.

If someone has antibodies to a viral hemagglutinin, then the serum of that person will inhibit agglutination by the virus to which they have antibodies - but not by other hemagglutinating viruses. This can be used to determine which hemagglutinating virus a person has been exposed to.

Hemadsorption relies similarly on the ability to bind red blood cells. If a virus puts a viral attachment protein into the plasma membrane of the infected cell which can bind to receptors on red blood cells, then the infected cell will bind red blood cells. This may enable virally infected cells to be detected at an early stage in infection, and may enable the detection of viruses which do not visibly damage the cell.

ADSORPTION AND PENETRATION

The H(N)/G protein recognizes receptors on cell surface.

The F protein facilitates fusion between membranes at physiological pH, so although paramyxoviruses can be taken up by coated pits, they also often enter the cell by direct fusion with the plasma membrane. Hence  syncytia can be formed in paramyxovirus infections.

Events inside the cell are very similar to rhabdoviruses:
Viral multiplication occurs in the cytoplasm.
RNA polymerase and RNA modification enzymes are packaged in the virion.
The RNA polymerase does not need a fully uncoated nucleocapsid.
Viral mRNAs are transcribed; these are capped, methylated and polyadenylated
The viral mRNAs are translated to give viral proteins.
There is no  distinction between early and late functions in gene expression.

Viral RNA replication involves full length plus strand synthesis. This is used as a template for full length minus strand. Both full length strands are coated with nucleocapsid protein as they are made.

New full length minus strands may serve as templates for replication, or templates for transcription, or they may be packaged into new virions.

ASSEMBLY

Both viral glycoproteins [i.e. attachment protein and F (fusion) protein] are translated as transmembrane proteins and transported to the cell plasma membrane.
M (matrix) protein enables nucleocapsids to interact with the regions of the plasma membrane which have the glycoproteins inserted.
Virus buds out through membrane.

In those paramyxoviruses which have it, the neuraminidase may facilitate release. In these viruses, sialic acid appears to be an important part of the receptor. The neuraminidase removes sialic acid (neuraminic acid) from the cell surface. Thus, since sialic acid will have been largely removed from the cell surface and the progeny virions, neither will have functional receptors, so progeny virions will not stick to each other or to the cell they have just budded out from (or any other infected cell). They will therefore be able to diffuse away until they meet an uninfected cell.

The neuraminidase may also help during infection since, if the virus binds to sialic acid residues in mucus, it would not be able to bind to a receptor on a cell and infect that cell. But if the sialic acid in the mucus is eventually destroyed, the virus will be freed and may then reach a receptor on the cell surface.

ACTIVATION OF THE F PROTEIN

The F protein needs to be cleaved before it can function in facilitating fusion when the virus binds to another cell. This is a late event in maturation.

Orthomyxovirus (Influenza A) © Dr Linda Stannard, University of Cape Town, South Africa

Orthomyxovirus (Influenza A) © Dr Linda Stannard, University of Cape Town, South Africa

  Bunyavirus From ICTV database

Examples: 

Orthomyxoviruses
Bunyaviruses (include Hantavirus genus)
Arenaviruses

There are three groups of influenza virus: A, B and C.   Influenza A virus is most intensively studied.

Pleomorphic virions
Negative-sense, single-stranded RNA
RNA genome is SEGMENTED, eight segments in influenza A
Helical nucleocapsid
Virions contain RNA polymerase
Enveloped, two membrane glycoproteins:
        HA - hemagglutinin - attachment and fusion protein
        NA - neuraminidase - important in release

Influenza virus has an unusual mechanism for acquiring a methylated, capped  5'end to its mRNAs:

A viral endonuclease (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.

The viral RNA polymerase (transcriptase) copies the template into complementary plus sense mRNA and adds a poly(A) tail.

Products of transcription:
The virus makes 8 primary transcripts:  one per segment.

Two of these 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 mature mRNAs. Thus a single segment can code for more than one protein since the virus has access to splicing machinery.

mRNAs are translated in the cytoplasm. Transmembrane proteins are moved to the plasma membrane while proteins needed for RNA replication go to the nucleus.

REPLICATION OF RNA
RNA replication occurs in the nucleus using a virus coded enzyme (this may be same as the RNA polymerase involved in transcription of mRNAs, or a modified version).
A full length, exact complementary copy of virion RNA is made - this plus sense RNA is probably coated with nucleocapsid protein as it is made.
Full length plus strand RNA is then used as a template for full-length minus strand synthesis; again the new minus strand is probably coated with nucleocapsid protein as it is made.
New minus strands can be used as templates for replication, mRNA synthesis, or packaged.

ASSEMBLY
This occurs at the plasma membrane.
Nucleocapsids are transported out of the nucleus.
Envelope proteins are transported via the Golgi body to the plasma membrane.
The M protein is involved - it interacts with both nucleocapsid and modified region of plasma membrane which contains the glycoproteins HA and NA.
Virus buds out.

NOTE:  HA needs to be cleaved before it can promote fusion on the next round of infection. The requirement for cleavage affects which tissues can produce infectious virus.

NA probably helps the virus leave the cell by removing sialic acid from receptors. NA may also help the virus penetrate mucus to reach epithelial cells of respiratory tract by enabling it to dissociate from sialic acid-containing receptors in the mucus by destroying them.

(The virus can infect cells because endocytosis presumably occurs before the receptors can be destroyed.)

There are similarities and differences between the Paramyxovirus family and the Orthomyxovirus family, members of both are enveloped, both contain negative sense, single stranded RNA, have helical nucleocapsids. However, the two families are very different. There is NO immunological relationship between the two families.

Reovirus family include:

the members of the reovirus genus
the members of the rotavirus genus
the members of the orbivirus genus
Colorado tick fever virus

Icosahedral symmetry, multiple shelled capsid
 RNA is double stranded. There are 10-12 segments (depending on the genus of the Reovirus family to which the virus belongs)

There are some significant differences in the life cycle of members of the reovirus family and of the rotavirus family. Due to their clinical importance in humans, we shall focus on rotaviruses.

ROTAVIRUSES (rota = wheel)

ADSORPTION PENETRATION AND UNCOATING
It is still not clear what exactly what happens in vivo.
There appears to be a need for a protease  to generate an "intermediate sub-viral particle" (ISVP) before the virus can enter the cytoplasm.
In vivo, the ISVPs are probably generated by protease digestion in the GI tract.
A viral attachment protein, probably at the vertices, recognizes host cell receptors.
The activated (ISVP) enters the cytoplasm directly or via endocytosis.

TRANSCRIPTION AND TRANSLATION
Double stranded RNA does not function as an mRNA and so the initial step is to make mRNA (transcription).
The mRNAs are made by virion-packaged RNA polymerase. It is also capped and methylated by virion packaged enzymes. It is then extruded from the capsid.

RELEASE
Probably occurs via cell lysis.

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