Revised Oct 2007

Dr. Margaret Hunt 

VIROLOGY - CHAPTER  FOUR  

RNA VIRUS REPLICATION STRATEGIES    

VIDEO LECTURE

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)

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 THAT DO NOT HAVE A DNA PHASE

Viruses that replicate via RNA intermediates need an RNA-dependent RNA-polymerase to replicate their RNA, but animal cells do not seem to possess a suitable 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:

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 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, does not function as mRNA immediately on infection since it is not released from the capsid into the cytoplasm. Instead, it serves as a template for reverse transcriptase and is 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

iv. the viral mRNA has special features which enable ribosomes to bind internally instead of (or as well as) at the 5’ end

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.

Figure 1 Polio virus © 1999 Dr J-Y Sgro, University of Wisconsin Biotechnology Center, Molecular Graphics Resources. Used with permission

Figure 2 Polio virus x350,000 © Dennis Kunkel Microscopy, Inc.  Used with permission

Examples: 

• picornaviruses

• togaviruses

• flaviviruses

1. PICORNAVIRUSES   (PICORNAVIRIDAE)

PROPERTIES

These are small (28nm), naked icosahedral viruses (figure 1)  (pico=very small). The RNA is single-stranded, plus sense, polyadenylated. It functions as mRNA immediately upon infection
Prototype member: poliovirus (figure 1 and 2)

ADSORPTION AND PENETRATION

A viral protein recognizes a receptor on the host cell membrane (this is important in the tropism of virus).
It seems that binding to the receptor alters capsid structure in some way, a channel forms across the cell membrane and the RNA is released into cytoplasm. The 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" (known as the internal ribosome entry site or IRES) which enables ribosomes to bind without having to recognize a 5' methylated cap structure (figure 3).

Picornaviruses often interfere with host cell methylated cap recognition. Most host cell translation is cap-dependent, so this inhibits a lot of host protein synthesis but not viral protein synthesis - one way in which these viruses can modify the host cell to their advantage.

The mRNA is translated into a single polypeptide (polyprotein), which is cleaved. The cleavages occur before translation is complete ( i.e. on the nascent (=growing) chain) and are carried out by virally coded proteases (figure 4). Some of these proteases can work even while part of the polyprotein.

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)
Viral RNA polymerase (replicase)
Certain 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 (figure 5). Again, poliovirus RNA polymerase and VPg are needed. VPg is linked to the 5' ends of the new plus sense strands (again, it probably functions as a primer).

The 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 usually 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 as the virions mature and these mature virions are infectious. Virions are released following cell lysis. Excess capsids are formed and inclusion bodies may be seen in the cytoplasm.

Examples: 

• Rhabdoviruses (figure 6)

• Paramyxoviruses (figure 11)

Figure 7 Structure of a typical rhabdovirus

Figure 7b
Rabies virus budding from an inclusion (Negri body) into the endoplasmic reticulum in a nerve cell.  A. Negri body. B. Notice the abundant RNP in the inclusion. C. Budding rabies virus. CDC

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

RNA: 

• is single stranded

• is negative (minus) sense

• codes for 5 proteins

ATTACHMENT, PENETRATION AND UNCOATING
The virus adsorbs to cell surface.
G (Glycoprotein) is the attachment protein (figure 7) which  binds to a receptor on the host cell surface.
The attached virus is taken up by endocytosis.
The membrane of the virus fuses with the endosome membrane (the acid pH of endosome is important because the G protein needs to be exposed to acid pH before it can facilitate fusion ).
As a result of fusion of the viral membrane with the endosome membrane, 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 for transcription - 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 (figure 8). 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

Messenger RNAs are translated on host ribosomes and all five viral proteins made at the same time. 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 (figure 8).
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 synthesizes  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, 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 (figure 9).

Nucleocapsid

Synthesis of the nucleocapsid was described above. The viral RNA polymerase complex associates with the nucleocapsids as they are formed. Nucleocapsids bud out through modified areas of membrane which contain G and M proteins (figure 10). The M (matrix) protein is involved in assembly - it interacts with patches of G in the membrane and with nucleocapsids.

NOTE:

• THE ENTIRE LIFE CYCLE OCCURS IN THE CYTOPLASM

• RNA POLYMERASE AND RNA MODIFICATION ENZYMES ARE VIRALLY-CODED AND PRESENT IN THE VIRION

  THERE IS NO EARLY/LATE DIVISION

Paramyxoviruses (figure 11) are pleomorphic, that is: there are many morphological forms of the virus in a population. They have negative-sense, non-segmented RNA and a helical nucleocapsid (figure 12). They are enveloped, that is they are surrounded by a membrane derived from a host cell.
The 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, the antibodies will binds to the attachment protein and prevent its binding to the red blood cells. The serum of that person will inhibit the agglutination reaction 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

During infection, the viral attachment protein will be inserted into the plasma membrane of the infected cell. If the viral attachment protein can bind to red blood cells, the infected cell will bind red blood cells because it has the viral attachment protein on its surface - this is called hemadsorption. In the clinical laboratory, this may enable virally-infected cells to be detected at an early stage in infection, and may allow 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 endocytosis, they also often enter the cell by direct fusion with the plasma membrane (figure 13).

Because the F protein functions at physiological pH, this can result in syncytia being formed in paramyxovirus infections (see discussion of consequences of fusion at physiological pH under DNA virus replication strategies – herpesviruses).

Events inside the cell are very similar to rhabdoviruses (figure 14):

• Viral multiplication occurs in the cytoplasm.

• The viral RNA polymerase uses the nucleocapsid as a template.

• The RNA polymerase does not need a fully uncoated nucleocapsid.

• Viral mRNAs are transcribed; these are capped, methylated and polyadenylated.

• Since this is a negative-strand RNA virus, RNA polymerase and RNA modification enzymes are packaged in the virion.

• 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 (figure 14).

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

Figure 15

Figure 16 Orthomyxovirus (Influenza A) © Dr Linda Stannard, University of Cape Town, South Africa

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.
The 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 (figure 15). This is a late event in maturation.

Figure 17 Orthomyxovirus (Influenza A) © Dr Linda Stannard, University of Cape Town, South Africa

Figure 18 Bunyavirus From ICTV database

Figure 19b   Vero E6 tissue culture cell infected with an arenavirus.  Image shows extracellular virus particles budding from the cell surface. Magnification approx. 12,000 times.   Image courtesy Cynthia Goldsmith, MS, Infectious Disease Pathology Activity, DVRD, NCID, CDC

Examples: 

• Orthomyxoviruses (figure 16 and 17)

• Bunyaviruses (include Hantavirus genus) (figure 18)

• Arenaviruses (figure 19b)

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. They have negative-sense, single-stranded RNA and an RNA genome that is SEGMENTED. There are eight RNA segments in influenza A. The nucleocapsid is helical (figure 19). Virions contain RNA polymerase packaged within the virus particle

These viruses are enveloped and have two 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

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 (figure 20) - hence all flu mRNAs have a short stretch at the 5' end which is derived from host mRNA.

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 (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 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.

NOTE:  

• HA needs to be cleaved before it can promote fusion. Cleavage occurs as the virus leaves the cell or in the extracellular fluid. The requirement for cleavage affects which tissues can produce infectious virus. The cleaved protein needs to then undergo a conformational change, usually caused by exposure to a acidic endosome environment when it infects the next cell, before it can cause fusion.

• 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 the respiratory tract by enabling it to dissociate from sialic acid-containing receptors in the mucus by destroying them. The neuraminidase does not prevent the virus infecting new cells because endocytosis is presumably faster than receptor removal.

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.

The 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

Reoviruses have icosahedral symmetry and a multiple layered capsid (inner and outer capsid) (figure 22)
The RNA is double stranded. There are 10-12 segments (depending on the genus of the Reovirus family to which the virus belongs) (figure 22).

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 (from appearance of virions in the electron-microscope)) (figure 23)

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 remove some of the outer layer of the capsid and 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 is then exposed on the ISVP, probably at the vertices, and binds to host cell receptors. The activated ISVP enters the cytoplasm directly or via endocytosis. In the cytoplasm, the virion RNA is copied by the viral RNA polymerase while still in a nucleocapsid that has fewer proteins associated with it than are associated with the ISVP or the virion.

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 virally-coded RNA polymerase packaged in the virion. The RNA is capped and methylated by virion packaged enzymes. It is then extruded from the vertices of the capsid.

ASSEMBLY
More proteins are made and eventually the immature capsids bud into the lumen of the endoplasmic reticulum. In doing so, they acquire a transient envelope which is lost as they mature. This is a very odd feature of the rotaviruses.

RELEASE
This probably occurs via cell lysis.

NOTE: THE ENTIRE REPLICATION CYCLE OCCURS IN THE CYTOPLASM

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