Picornavirus replication

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Picornaviruses are genetically simple and have been the subject of extensive experimental investigation owing to the number of diseases they cause. Their name is based on a pseudoclassical use of Latin mixed with modern terminology: pico ("small")-RNA-virus.

The replication of poliovirus (the best-characterized picornavirus, and perhaps, best-characterized animal virus) provides a basic model for RNA virus replication. Studies on poliovirus were initiated because of the drive to develop a useful vaccine against paralytic poliomyelitis. These studies successfully culminated in the late 1950s and early 1960s. Protocols developed for replicating the virus in cultured cells formed the basis for successful vaccine development and production. At the same time, the relative ease of maintaining the virus and replicating it in culture led to its early exploitation for molecular biological studies. It is still a favored model.

Other closely related picornaviruses include rhinoviruses and hepatitis A virus. These replicate in a generally similar manner, as do a number of positive-sense RNA-containing bacterial and plant viruses. Indeed, close genetic relationships among many of these viruses are well established.

The poliovirus genetic map and expression of poliovirus proteins

A schematic of the icosahedral poliovirus virion is shown in Fig. 14.2. In accordance with its classification as a positive-sense RNA virus, the poliovirus genomic RNA isolated from purified virions is mRNA sense and acts as a viral mRNA upon infection. Full characterization and sequence analysis has established that the genome is 7741 bases long with a very long (743-base) leader sequence between the 5' end of the mRNA and the (ninth!) AUG, which initiates the beginning of an ORF extending to a translation termination signal near the 3' end. There is a short untranslated trailer following the 7000-base ORF, and this is followed by a polyA tract. The polyA tail of the poliovirus mRNA is actually part of the viral genome; therefore, it is not added posttranscriptionally as with cellular mRNA (see Chapter 13). A simple genetic map of the viral genome is shown in Fig. 14.2.

Recently, the entire genome of poliovirus was assembled from oligodeoxynucleotides as a double-stranded cDNA molecule and subsequently transcribed by RNA polymerase into infectious RNA. The experiment, reported in 2002, raised some issues of security with respect to possible bioterrorist implications of this work. In fact, the synthesis of infectious influenza virus, using preserved tissue material from the extremely virulent 1918 strain, led to an even stronger reaction. These issues highlight the increasingly sensitive nature of some aspects of modern virological research.

While poliovirus RNA is mRNA and can be translated into protein in an in vitro translation system, it has two properties quite different from cellular mRNA. First, poliovirus virion RNA has a protein VPg at its 5' end instead of the methylated cap structure found in cellular mRNA. The VPg protein is encoded by the virus. The viral mRNA also has a very long leader that can assume a complex structure by virtue of intramolecular base pairing in solution. The structure of this leader sequence, especially near the beginning of the translational reading frame [the internal ribosome entry site (IRES)], mediates association of the viral genome with ribosomes. The IRES structure and its role in translation initiation is an alternate way in which eukaryotic ribosomes can initiate protein synthesis without binding at the 5' end and transiting to an AUG codon. Subsequent to the characterization of its role in picornavirus replication, it has been

Pentamer

RNA core (VpG)

Capsid

Pentamer

Poliovirus

Picornavirus

Capsid

Poliovirus

743 nt

Single ORF (~7000 nt)

Translate P-1 precapsid P-2

Autocleavage P-2

VP0 VP3

2B/C

3A/B

3C/D

2A 2B 2C

(2A protein) 3A VpG 3C 3D

VP4 VP2 VP3 VP1

Capsid

Fig. 14.2 (a) Poliovirus, a typical picornavirus. The 30-nm-diameter icosahedral capsid comprises 60 identical subunits — each a pentamer of subunits (often called protomers) containing a single copy of VP1, VP2, VP3, and VP4. The map of the approximately 7700-nucleotide (nt) single-stranded RNA genome that serves as mRNA in the initial stages of replication is also shown. Unlike cellular mRNA, poliovirus genomic RNA has a viral protein (VPg) at its 5' end instead of a methylated nucleotide cap structure. The RNA has a c. 740-nt sequence at the 5' end that encodes no protein, but assumes a complex secondary structure to aid ribosome entry and initiation of the single translational reading frame. The single precursor protein synthesized from the virion RNA is cleaved by internal proteases (2A and 3C) initially into three precursor proteins, P1, P2, and P3. Protein P1 is then proteolytically cleaved in a number of steps into the proteins that assemble into the precapsid, VP0, VP1, and VP3. Proteins P2 and P3 are processed into replicase, VPg, and a number of proteins that modify the host cell, ultimately leading to cell lysis. With three exceptions, all proteolytic steps are accomplished by protease 3C, either by itself or in association with protein 3D. Protease 2A carries out the first cleavage of the precursor protein into P1 and P2 as an intramolecular event. It also mediates cleavage of the protease 3CD precursor into protease 3C and protein 3D. It is not known how the third cleavage that does not utilize protease 3C occurs. This is the maturation of the capsomers by the cleavage of VP0 into VP2 and VP4. The VP4 protein is modified by the addition of a myroistyl residue at the amino terminus (myr = myroistyl). (b) The structure of the poliovirus internal ribosome entry site (IRES). The diagram is a schematic of the predicted secondary structure in the 5' proximal region of the poliovirus genome. The shaded secondary structure features make up the IRES. Note that one of the mutations associated with attenuation of the Sabin vaccine strains is located in this region. The site of the AUG at which initiation of the large polyprotein occurs is also indicated.

  • b) Internal ribosome entry site (IRES)
  • b) Internal ribosome entry site (IRES)
Sabin Ncr Poliovirus Structure

Fig. 14.2 Continued found to function in the translation of several cellular transcripts also. With poliovirus RNA, the normal Kozak rules for the selection of the AUG codon to initiate translation in an mRNA (see Chapter 13, Part III) do not apply. Indeed, the AUG triplet that begins the large poliovirus ORF is preceded by eight other AUG triplets within the leader that are not utilized to initiate translation. The structure of this region of the poliovirus RNA genome is shown in diagrammatic form in Fig. 14.2(a). The IRES structure is now used routinely in the construction of plasmids where an internal ribosome initiation is needed.

Upon successful initiation of infection, viral genomic mRNA is translated into a single large protein that is the precursor to all viral proteins. This precursor protein is also shown in Fig. 14.2(b); it contains all the poliovirus proteins that are expressed during infection. Thus, all the viral proteins such as those shown in Fig. 12.1 are derived from it.

The smaller proteins are cleaved from the precursor polyprotein by means of two proteases (2A and 3C) that comprise part of this large viral protein. As briefly outlined in Chapter 6, Part II, many viruses utilize proteolytic cleavage of large precursor proteins via virus-encoded proteases during the replication process, and such proteases are important potential targets for antiviral chemotherapy (see Chapter 8). Indeed, the development of protease inhibitors has had a very encouraging effect on attempts to treat AIDS.

The steps in processing are complex, and have yet to be fully worked out in complete detail. Both viral proteases utilize a cysteine residue as part of their active sites; thus, they are termed C-proteases. They exhibit a very high specificity, and although both cleave the precursor peptide at sites between specific amino acids (Tyr-Gly for protease 2A and Gln-Gly for protease 3C), neither cleaves all available sites and protease 2A does not cleave nonviral peptides with any efficiency at all. Clearly, secondary structure and other features of the substrate protein are important in determining cleavage sites.

The first two cleavages take place intramolecularly, that is, within the protein in which the proteases are covalently linked. These cleavages result in the formation of three large precursor proteins, P1, P2, and P3. Protein P1 contains the capsid proteins, VP1, VP3, and VP0, as well as a short leader protein (L). While not established for poliovirus, the L protein of other picor-naviruses has been associated with both virus assembly as well as cellular trafficking pathways. In addition, the P1 protein is myristoylated at the N-terminal end, involving the covalent addition of the 14-carbon fatty acid myristic acid. As a result, the N-terminus of VP0 will have this modification, which is known to enable such modified proteins to associate efficiently with membrane structures. The P2 and P3 proteins are precursors for a number of nonstructural proteins, including the viral replicase enzyme and proteins and enzymes that alter the structure of the infected cell. Protein P3 also contains the VPg protein. The general steps in derivation of mature viral proteins from the precursor protein are shown in the genetic map of Fig. 14.2(a).

The later stages in processing of the precursor proteins involve mainly protease 3C, although protease 2A cleaves the 3CD precursor of protease and replicase into variants then termed 3C' and 3D'. It remains unknown whether these variants have any role in replication, given that they are not seen in infections with all strains of the virus. While protein 3D is not a protease (it is the replicase protein), it aids in cleavage of the VP0-VP3 precursor into VP0 and VP3. The 3CD precursor itself, however, can also act as a protease, and may have a specific role in some of the early cleavage events.

Since the poliovirus ORF is translated as a single, very large protein, poliovirus technically has only one "gene." This is not strictly true, however, since different portions of the ORF contain information for different types of protein or enzyme activities. Further, different steps in processing of the precursor proteins are favored at different times in the replication cycle; therefore, the pattern of poliovirus proteins seen varies with time following infection, as shown earlier in Fig. 12.1.

The demonstration of precursor—product relationships between viral proteins can be tricky and experimentally difficult, but the procedure's theory is simple and based on analysis of proteins encoded by the virus, consideration of the virus's genetic capacity to encode proteins, and a general understanding of the translation process itself. The separation and enumeration of viral proteins based on their migration rates in denaturing gels, which is a function of protein size, are outlined in Chapter 12, Part III.

For poliovirus, many years of analysis can be summarized as follows: The total molecular size of the proteins encoded by the virus cannot exceed approximately 2300 amino acids (7000/3). Despite this, the total size of viral proteins estimated by adding radioactive amino acids to an infected cell and then performing size fractionation on the resulting radiolabeled material is significantly greater. Further, it is known that poliovirus efficiently inhibits cellular protein synthesis, so most proteins detected by the addition of radioactive precursor amino acids to infected cells (also termed a pulse of radioactive material) are, indeed, viral.

This conundrum can be resolved by using a technique called a pulse-chase experiment, and by using amino acid analogues, which inhibit protease processing of the precursor proteins. In pulse-chase experiments, radioactive amino acids are added for a short time. This is the "pulse." Then a large excess of nonradioactive amino acids is added to dilute the label. This is the "chase."

Only the largest viral proteins isolated from a poliovirus-infected cell exposed only to the radioactive pulse for short periods (followed by isolation of the infected cell) had radioactivity. This finding suggests that these proteins are the first viral products synthesized. If the pulse period is followed by chase periods of various lengths, radioactivity is eventually seen in the smaller viral proteins. Such a result is fully consistent with a kinetic precursor—product relationship between large (precursor) proteins and smaller mature (product) viral proteins.

The relationship between precursor and product was confirmed by adding translation inhibitors at specific times following a pulse of radioactive amino acids. This step resulted in the loss of label incorporated into large proteins, but did not affect the appearance of label in the smaller proteins derived from the precursor proteins already labeled during the pulse. Finally, addition of amino acid analogues that inhibited proteolysis of the precursor protein contributed a further confirmation of the process.

The poliovirus replication cycle

As shown in Fig. 14.3, everything tends to "happen at once" during the poliovirus replication cycle. Viral entry involves attachment of the virions by association with the cellular receptor.

Poliovirus Replication Cycle
  1. 14.3 The poliovirus replication cycle. The schematic representation is broken into discrete steps. Viral entry is by receptor-mediated endocytosis during which the virion proteins are sequentially removed, releasing virion-associated positive-sense RNA. Viral entry involves attachment to a cell surface receptor, followed by rearrangement of the capsid with insertion of the myroistylated-VP4 terminus into the cell membrane, releasing the positive-sense RNA genome into the cytoplasm of the cell. This RNA is translated into a large polyprotein. Viral replicase released from the precursor protein then mediates generation of RI-1 and RI-2 to generate more mRNA that, unlike the original genomic RNA, has the VPg protein cleaved off. As infection proceeds, the replication complexes become associated with cellular membrane structures into replication compartments. Newly synthesized positive-sense RNA is also translated and the process repeats many times until sufficient capsid protein precursors are formed to allow assembly of the procapsid. Procapsids associate with newly synthesized positive-sense RNA still containing VPg at its 5' end, and entry of viral genomes results in capsid maturation. As the process continues, virions accumulate in the cytoplasm until viral proteins induce cell lysis and virus release occurs. The entire process can take place in the absence of a nucleus.
  2. 14.3 The poliovirus replication cycle. The schematic representation is broken into discrete steps. Viral entry is by receptor-mediated endocytosis during which the virion proteins are sequentially removed, releasing virion-associated positive-sense RNA. Viral entry involves attachment to a cell surface receptor, followed by rearrangement of the capsid with insertion of the myroistylated-VP4 terminus into the cell membrane, releasing the positive-sense RNA genome into the cytoplasm of the cell. This RNA is translated into a large polyprotein. Viral replicase released from the precursor protein then mediates generation of RI-1 and RI-2 to generate more mRNA that, unlike the original genomic RNA, has the VPg protein cleaved off. As infection proceeds, the replication complexes become associated with cellular membrane structures into replication compartments. Newly synthesized positive-sense RNA is also translated and the process repeats many times until sufficient capsid protein precursors are formed to allow assembly of the procapsid. Procapsids associate with newly synthesized positive-sense RNA still containing VPg at its 5' end, and entry of viral genomes results in capsid maturation. As the process continues, virions accumulate in the cytoplasm until viral proteins induce cell lysis and virus release occurs. The entire process can take place in the absence of a nucleus.

For poliovirus the receptor, Pvr, is a specific CAM-like molecule (CAM = cellular adhesion molecule) called CD155. The binding of poliovirus virions to the receptor has been examined by x-ray crystallography and appears to involve insertion of a part of the receptor into "canyon" cavities on the surface of the virus particle.

Since poliovirus is able to efficiently infect cells that are mutated in the protein dynamin, required for the function of clathrin-coated pits, it is now thought that poliovirus does not enter host cells by way of receptor-mediated endocytosis, as diagrammed in Fig. 6.2, even though some other picornaviruses may depend upon this pathway of entry. The current model for poliovirus attachment and entry into the cell is as follows (Fig. 14.3):

1 Virus particles attach to Pvr, the poliovirus receptor on the surface of the cell.

2 Receptor binding induces a rearrangement of the virus particle that results in the insertion of helical regions of VP1 into the cell membrane, along with the myristoylated amino terminal end of VP4, thus creating a channel into the cytoplasm.

3 Viral RNA is released into the cell cytoplasm after further particle rearrangements, perhaps triggered by ionic changes.

Viral RNA is translated into protein, portions of which are involved in replication of the viral genome by generation of the replication structures, RI-1 and RI-2. The protein VPg is a primer for this replication by having a uracil residue added to it, a process called uridylation. The initiation of replication requires a RNA secondary structure feature called the cis-acting replication element (CRE), located within the coding region in the genome for the 2C protein. Poliovirus replicase, protein 3Dp°', catalyzes the generation of both negative- and positive-sense products. It has recently been demonstrated that cis-acting sequence elements that control replication are present in the poliovirus genome. Secondary structure features at the 5' end as well as within coding regions appear to be required for efficient RNA replication. Other poliovirus proteins are also involved, as well as one or more host proteins, since much of the viral genome's replication takes place in membrane-associated compartments generated by these proteins within the infected cell's cytoplasm. Generation of new mRNA sense (positive) strands of poliovirus RNA leads to further translation, further replication, and finally, capsid assembly and cell lysis.

Details of the poliovirus capsid's morphogenesis were worked out several decades ago. While there is still some controversy concerning the timing of certain steps in the assembly process (especially the timing of the association of virion RNA with the procapsids), poliovirus assembly serves as a model for such processes in all icosahedral RNA viruses (see Chapter 6, Part II). Proteolytic cleavage of precursor proteins plays an important role in the final steps of maturation of the capsid. This cleavage does not involve the action of either protease 2A or 3C. Rather, it appears to be an intramolecular event mediated by the capsid proteins themselves as they assemble and assume their mature conformation. The molecular sizes of the poliovirus capsid proteins are given in Table 11.1.

The most generally accepted scheme is shown in Fig. 14.4. In viral morphogenesis, myris-toylated-Pl protein is cleaved from the precursor protein by protease 2A segment. Five copies of this protein aggregate and the protein is further cleaved by protease 3C into myristoylated-VP0, VP1, and VP3, which forms one of the 60 capsid protomers. Five of these protomers assemble to form the 14s pentamer. Finally, 12 of these 14s pentamers assemble to form an empty capsid (procapsid).

This procapsid is less dense than the mature virion, so its proteins can be separated readily by centrifugation. Analysis of the procapsid proteins demonstrates equimolar quantities of myristoylated-VP0, VP1, and VP3. Following formation of the procapsid, viral RNA associates with the particle and a final cleavage of VP0 into VP2 and myristoylated-VP4 occurs to generate the mature virion. After virions are assembled, the cell lyses and virus is released

Picornavirus cytopathology and disease

The most obvious cytopathology of poliovirus replication is cell lysis. But prior to this, the virus specifically inhibits host cell protein synthesis. Inhibition of host cell protein synthesis involves proteolytic digestion of the translation initiation factor eIF-4G so that ribosomes can no longer recognize capped mRNA (see Chapter 13, Part III). Such modification leads to the translation of only uncapped poliovirus mRNA because its IRES allows it to assemble the translation complex with the virus-modified ribosomes. Note that this rather elegant method of shutoff will not work with most types of viruses because they express and utilize capped mRNA!

P1 precapsid

Nascent cleavage by 2A protease segment P2

N Myr

Myrc

Cleavage by 3C protease segment VP0 VP3 VP1

Maturation VP1^^VP3

Protomer (5s)

Procapsid (73s)

Procapsid (73s)

Pentamer (14s)

RNA encapsidation

Provirion (155s)

Cleavage of VP0 into VP2 + VP4

Fig. 14.4 The steps in the assembly of the poliovirus virion. Precursor proteins associate to form 5s protomers, which then assemble to form pentamers. Twelve of these assemble to form the procapsid into which virion RNA is incorporated. Final cleavage of VP0 into VP2 and VP4 takes place to form the mature capsid that has a diameter of 28—30 nm.

Virion (155s)

Virion (155s)

There are three related types, or serotypes, of poliovirus. They differ in the particular antigenic properties of viral structural proteins. Most poliovirus infections in unprotected human populations result in no or only mild symptoms, but one serotype (type 3) is strongly associated with the disease's paralytic form. Infection with this serotype does not invariably lead to a paralytic episode, but the probability of such an episode is much higher than with the others. All serotypes are distributed throughout the regions where poliovirus is endemic in a population, although some predominate in some locations.

Poliovirus is spread by fecal contamination of food or water supplies. Receptors for the virus are found in the intestine's epithelium, and infection results in local destruction of some tissue in the intestine, which can result in diarrhea. Unfortunately, motor neurons also have receptors for poliovirus, and if the virus gets into the bloodstream, it can replicate in and destroy such neurons, leading to paralysis. This result is of no value to the virus since the virus initiating neuronal infection cannot be spread to other individuals and is eventually cleared; thus, the paralytic phase of the disease is a "dead end" for the virus. The virus stimulates an immune response and the individual recovers and is resistant or immune to later infection.

Vaccination against poliovirus infections is accomplished effectively with both inactivated and attenuated live-virus vaccines, as described in Chapter 8, Part II. Since the only reservoir of poliovirus is humans, immunity through vaccination against the virus is an effective way of preventing disease. Currently, a major effort is underway to completely eradicate the disease from the environment (see Chapter 25, Part V).

A number of other picornaviruses cause disease; many are spread by fecal contamination and include hepatitis A virus, echoviruses, and coxsackievirus. Like poliovirus, these viruses occasionally invade nervous tissue. Coxsackievirus generally causes asymptomatic infections or mild lesions in oral and intestinal mucosa, but can cause encephalitis. Echoviruses are associated with enteric infections also, but certain echovirus serotypes cause infant nonbacterial meningitis, and some epidemic outbreaks with high mortality rates in infants have been reported.

Another widespread group of picornaviruses are the rhinoviruses, one of the two major groups of viruses causing common head colds. Unlike the other picornaviruses detailed here, rhinoviruses are transmitted as aerosols. Because of the large number (~100) of distinct serotypes of rhinovirus it is improbable that an infection will generate immunity that prevents subsequent colds. There are no known neurological complications arising from rhinovirus infections.

Flavivirus replication

The success and widespread distribution of picornaviruses and their relatives demonstrate that the replication strategy found in translation of a single large ORF is a very effective one. If more evidence were needed on this score, the plethora of mosquito-borne flaviviruses should settle the matter completely!

Flaviviruses are enveloped, icosahedral, positive-sense RNA viruses. They appear to be related to picornaviruses, but clearly have distinct features, notably an envelope. Because mosquitoes and most other arthropods are sensitive to weather extremes, it is not surprising that arboviral diseases occur throughout the year in the tropics and subtropics, but occur only sporadically, and in the summer, in temperate zones.

Many flaviviruses demonstrate tropism for neural tissue, and flaviviruses are the causative agents of yellow fever, dengue fever, and many types of encephalitis. In the United States, mosquito-borne St Louis encephalitis virus leads to periodic epidemics in the summer, especially during summers marked by heavy rains and flooding, such as the summer of 1997 in northeastern states.

West Nile virus was first isolated in the Middle East, as suggested by its name. However, it has recently invaded the western hemisphere and is firmly established throughout the United States. The scenario began in the late summer of 1999, when at least 1900 people in Queens, New York City, were infected with West Nile. Analysis of the virus suggested that it originated from a strain present in Israel. No one knows how this virus arrived in New York. However, it soon spread into the wild bird population and began its march across the country. As of the end of 2005, the virus is present in all of the contiguous states, with most reporting both human and animal cases. We can now say that West Nile virus has established itself as endemic in North America.

Fig. 14.5 The yellow fever virus (a flavivirus) and its genome. This flavivirus has a replication cycle very similar in broad outline to that detailed for poliovirus. Unlike poliovirus, flaviviruses encode a single envelope glycoprotein, and its approximately 10,000-nucleotide (nt) genome is capped, although not polyadenylated. Also in contrast to poliovirus, the yellow fever virus precursor polyprotein is cleaved into a large number of products as it is being translated, so the very large precursor proteins of poliovirus replication are not seen. The enveloped capsid is larger than that of poliovirus, with a diameter of 40—50 nm. ER = endoplasmic reticulum.

Yellow fever virus (flavivirus)

Yellow fever virus (flavivirus)

Envelope

Lipid bilayer Nucleocapsid

Single ORF (10,233 nt)

Envelope

Lipid bilayer Nucleocapsid

118 nt

Single ORF (10,233 nt)

51 nt

Translation Proteolytic cleavage

Capsid pre M

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