The genomes of positive strand RNA viruses often contain more than one open reading frame. Some of these viruses have evolved novel mechanisms to regulate the synthesis of the other open reading frames that in some cases involved the production of a subgenomic RNA or RNAs. Very often, the presence of the subgenomic RNA is used as indicator for active viral genome replication. Norovirus, a major cause for gastroenteritis as well as with all other caliciviruses follow a typical positive strand RNA viruses genome replication strategy. In addition, noroviruses also produce a subgenomic RNA during their replication in infected cells. Efficient and adequate synthesis of norovirus subgenomic RNA is crucial for successful viral replication and productive infection leading to the generation of infectious viral progeny. This chapter will dissect the significant findings on mechanisms involved in norovirus genome replication as well as focusing on subgenomic RNA production.
- RNA-dependent RNA polymerase
- subgenomic RNA
- internal initiation
- core promoter
Noroviruses are often associated with outbreaks of gastroenteritis in hospitals, on cruise ships, schools, nursing homes and military camps where a close person to person contact cannot be avoided . Infection is typically followed by a 24 to 48 hour incubation period before emergence of the clinical disease, symptoms of which include acute diarrhea and projectile vomiting, usually accompanied by several signs/symptoms such as abdominal cramps, myalgia, malaise, headache, nausea and low grade fever [2, 3]. Noroviruses are the most common cause of gastroenteritis infections due to their stability, low infectious dose, large host reservoir (humans), short term immunity, multiple transmission routes and large genetic diversity between strain . The human norovirus (HuNv) infection is self-limiting and the symptoms typically last between 12 and 60 hours . However, viral shedding appears to be prolonged up to several weeks after the symptoms are resolved, especially in persons with impaired immunity where persistent infection often occur by reinfection [5, 6]. More importantly, illness among the elderly and immunocompromised patient can be fatal due to the severe dehydration. The main transmission route for noroviruses is by fecal-oral, through the contaminated food, water or surfaces especially [1, 7]. Consumption of contaminated fresh produce food such as salads, fruits and sandwiches that requires no prior heating have also been linked as a possible source of food-borne infections . Furthermore, a high concentration of norovirus was also found within the gastrointestinal tissue of contaminated bivalves such as oysters and mussels that are filter feeders. Therefore, these contaminated bivalves are also considered as another important foodborne source of norovirus infection . In addition, airborne transmission that involves the aerosolized vomit from an infected person has also been demonstrated [10, 11]. These findings are supported by the low infectious dose required for norovirus infection; less than 10 viral particles are sufficient enough to establish infection with Norwalk virus . First described as Norwalk virus, which was responsible for a gastroenteritis outbreak at a school in Norwalk, Ohio US, in 1968 , human noroviruses (HuNv) are today recognized as the leading cause of viral gastroenteritis infections in human population. The United States Centre for Disease Control and Prevention (CDC) has estimated that noroviruses are responsible for at least 23 million cases of food borne illness each year in the United States with approximately 50 thousands hospitalization and 300 death [4, 14]. However, in one of the reviews which involves a period of study from 1996 to 2007, it is estimated to be nearly 110,000 hospitalization per epidemic years with the cost of approximately 500 million US dollar per year . The recorded surveillance data from the Food Borne Viruses in Europe Network also indicates that more than 85% of viral gastroenteritis outbreaks that occurred between 1995 and 2000 could be attributed to these viruses [4, 16]. The cost to the United Kingdom National Health Service (NHS) in England and Wales as a direct result of the outbreaks occurring in hospitals has been estimated to be approximately 115 million pounds in 2002–2003 . However, due to the acute nature of the infection, it is difficult to identify all the norovirus infection cases and therefore the real cost can be considered higher. Furthermore, the global impact of gastroenteritis caused by HuNv is hard to be estimated since most of the annually 3.5 to 5 million deaths are from developing world with inadequate healthcare, surveillance and diagnostic systems . There is still no licensed vaccine against norovirus made available. However, there are few promising candidates in the pipeline with one already in phase 2 . In addition, efforts in developing norovirus-specific antiviral drugs are also ongoing. To enable these efforts, our fundamental knowledge on norovirus biology needs to be enhanced especially with regards to norovirus genome replication. This chapter will emphasize on subgenomic RNA replication aspect of norovirus particularly focusing on works with MNV.
2. Building of norovirus particle
The first norovirus virion to be observed by immune-electron microscopy was Norwalk virus in 1972 by Albert Kapikian. The virions are icosahedral, with a diameter ranging from 27 to 39 nm and a buoyant density of 1.36 ± 0.04 g/cm3 [4, 13, 19]. The virus’s capsids are composed of 180 copies of a major protein VP1 (formed into 90 dimers) and one or two copies of the minor capsid protein VP2 [20, 21]. Studies using Norwalk virus-like particles (VLPs) revealed that the major protein VP1 is structurally divided into two domains referred to as the ‘shell’ (S) and ‘protruding’ (P) domains, with the P domain being further divided into P1 and P2 subdomains . The inner S domain sub-units interact each other to form a continuous ‘shell’ structure for capsid while the P domain emanates from the S domain surface and forming cup-like structure. Furthermore, the outer P2 subdomain has been recognized as the most variable region of the calicivirus capsid and the region that determines the species-specific binding of these viruses to the respective cell receptor [22, 23]. Molecularly, norovirus particle capsid encloses the viral genome, a positive-sense single stranded RNA molecule of about 7.4 to 8.3 kb in size. The genome has a virus encoded protein covalently linked to the 5′ end (VPg) and a poly A tail at the 3′ end (Figure 1). This genomic RNA (G RNA) encodes three open reading frames (ORFs) flanked by two short untranslated regions (UTRs) and with a small degree of overlap at the 5′ and 3′ junctions between ORF1 and ORF2 [24, 25]. In addition, within the
3. The norovirus life cycle
Like all viruses, the life cycle of noroviruses begins with the attachment of the viral particles to their specific receptor on the membrane of the host cell. Susceptibility to norovirus infections in humans, specifically Norwalk virus, is associated with ABO histo-blood group antigens (HBGA) and individual secretor status . HBGA are carbohydrates found on the surface of gut epithelial cells [4, 20, 27]. These carbohydrate molecules are involved in the attachment of noroviruses but are unlikely to be the main receptor as co-receptor may also be involved . In addition, the secretor status of individuals also determines the susceptibility to norovirus infections . Individuals who are non-secretors of H type 1 were found to be resistant to norovirus infections due to a mutation in the α-(1,2)-fucosyltransferase (
After entry of the positive sense viral genome into the host cell cytoplasm, it can immediately act as mRNA for protein synthesis. The subsequent event of norovirus life cyle is a pioneer round of viral proteins translation from the positive strand viral genomic RNA. The norovirus VPg (viral protein genome link) protein is a 13–15 kDa non-structural protein covalently linked to the 5′ end of the viral genomic (G RNA) and subgenomic RNA (SG RNA) and acts as a cap substitute (Figure 1). The VPg protein recruits host cells translation initiation factors in initiating the translation process to produce viral proteins [36, 37]. This mechanism is a unique strategy employed by noroviruses to ensure the preferential translation of their RNA over host cell mRNA which possess a classical 5′ cap structure. In fact, all caliciviruses use this translational strategy since their 5’UTR is relatively short (only 5 nucleotides in MNV) compared to the closely related picornavirus genome which contains a much longer 5’UTR. Even though the picornavirus genome also possesses a VPg at the 5’end, this smaller protein (~22 amino acids) does not have any sequence homology with the calicivirus VPg and is not involved in picornavirus translation. Indeed, picornavirus translation is driven by the presence of an internal ribosomal entry site (IRES) structure within its 5’UTR . Translation of the first open reading frame of noroviruses typically yields a large polyprotein, representing the non-structural proteins. This large polyprotein is subjected to further processing by the virus encoded 3C-like (3CL) protease at five specific protease cleavage sites yielding six mature forms of the non-structural proteins [39, 40]. Sosnovtsev
The NS1/2 protein is the first non-structural protein in noroviruses (Figure 1) and is predicted to have a similar function to the picornavirus 2B protein, which is involved in membrane rearrangement and results in a modification of membrane permeability . The enterovirus 2B protein which is a member of the
The norovirus NS3 protein is a nucleotide triphosphatase (NTPase) (Figure 1). A study using a human norovirus (Southampton virus) showed that NS3 has NTPase activity that functions to hydrolyse nucleotide triphosphate . In MNV infected cells, the NS3 has been shown to associate with the viral replication complex . In addition, the equivalent protein in FCV called p39, was found to co-localize with viral replication complexes suggesting a possible role in replication [41, 47].
Little is known about the NS4 protein. However, it is thought that NS4 may play a role in tissue culture adaptation of MNV since repeated passage of MNV-1 in RAW264.7 cells give rise to attenuated viruses in part caused by sequence changes in NS4 . Furthermore, NS4 is also thought to recruit VPg to membranous replication complexes during replication . Targeted mutations in poliovirus 3A, the NS4 equivalent, resulted in viruses defective in RNA synthesis  indicating that by analogy, the norovirus NS4 may also contribute to viral RNA synthesis.
The NS5 encodes the viral VPg protein that plays a multifunctional role in the viral life cycle. The main role of VPg has been identified to be in translation initiation. This 13–15 kDa protein is covalently linked to the 5′ end of the G RNA and SG RNA of caliciviruses . VPg has been shown to be essential for viral RNA infectivity as treatment of viral RNA with proteinase K rendered the viral RNA non-infectious .
The NS6 encodes the viral 3C-like protease and is thought to play a role in inhibition of cellular protein synthesis in infected cells.
The NS7 protein, located at the C-terminus the norovirus ORF 1, encodes the RNA-dependent RNA polymerase (RdRp), which is a key enzyme in viral replication. This protein will be elaborated further in the subsequent subsection of this chapter because it plays a major role in viral G RNA and SG RNA replication.
The ORF2 and ORF3 of noroviruses code the structural proteins VP1 and VP2 respectively. Both of these proteins are expressed from the viral VPg-linked SG RNA that is 3′ co-terminal with the G RNA (Figure 1). However, in lagoviruses, sapoviruses and neboviruses, the capsid protein may also be produced from the G RNA as the capsid genes for these viruses are in frame with ORF1 giving rise to a polyprotein that contains both the non-structural proteins and the major capsid protein [59, 60]. ORF2 of norovirus encodes the 58.9 kDa major capsid protein (VP1) and ORF3 encodes the 22.1 kDa minor capsid protein (VP2) . The expression of VP1 protein with or without co-expression of VP2 allows dimer formation that can be further assembled to produce VLPs in the absence of RNA genome [62, 63, 64]. Since the HuNv is currently not efficiently propagated in tissue culture, VLPs have been used to study a variety of virus-host interactions as they are morphologically and antigenically indistinguishable from real virus particles . In FCV, the capsid protein contains a leader peptide (leader capsid or LC) at its N terminus that is cleaved by p76 to give rise to the mature capsid protein VP1. The VP2 protein has been shown to stabilize and protect VLPs from proteolytic degradation when this protein is co-expressed with VP1 in the baculovirus system . The very basic character of VP2 suggests an interaction with nucleic acid and it may contribute to the encapsidation of the viral RNA. However, this hypothesis has yet to be examined and confirmed. Furthermore, at least for FCV, the VP2 protein is essential for the production of infectious particles and for virus replication . In addition to the ORF2 and ORF3, there is another alternative ORF, namely ORF4, which was found in MNV, overlapping with the VP1 coding region and encoding the virulence factor 1 protein (VF1) . This VF1 protein has been demonstrated to play a role in infection and virulence
The pioneer round of viral proteins production is proceeded with G and SG RNA replication once the viral replication-related proteins are made available in the infected cell’s cytoplasm. This particular process will be further elaborated in separate section below. When all the viral proteins become available and the replication has occurred, the viral RNA progeny is then packaged into viral particles. As mentioned earlier, the VP2 protein may contribute to this event. The mechanism of calicivirus encapsidation has yet to be studied in great detail. Present evidence suggests that the SG RNA could be encapsidated separately in the case of RHDV as well as in FCV [68, 69]. However, little is known about the mechanisms of viral release, but since norovirus infections induce apoptosis, it is speculated that apoptosis-induced membrane collapse releases the virus particles from the infected cells [70, 71, 72].
4. The norovirus genome replication
Once the translation of the norovirus non-structural proteins has begun, their presence in infected cells induces the formation of cytoplasmic membrane-bound replication complexes, enabling the viral genome replication process to take place . These replication complexes, which contain the viral RdRp, viral RNA (single and double-stranded intermediates) and other viral enzymes and host cell factors, act as a surface or platform for the viral replication. The rearrangement of intracellular membranes (particularly the endoplasmic reticulum and Golgi apparatus) of MNV-1 infected RAW264.7 cells has been observed whereby membrane vesicles start to appear at twelve hours post infection . The elaboration of norovirus genome replication in this chapter will be done interchangeably with the function of the central replication enzyme, RdRp.
The RdRp, also known as the RNA replicase, is an enzyme that catalyzes the synthesis of RNA from RNA templates. This particular virus enzyme is therefore distinct from the typical eukaryotic DNA-dependent RNA polymerase that catalyzes transcription of mRNA from a DNA template. All RNA viruses carry an RdRp gene in their RNA genome since this viral replication enzyme is pivotal for genome replication in infected cells. In addition, the virions of negative strand and double-stranded genome viruses must contain the RdRp as a ribonucleoprotein component since the incoming RNA genome cannot be translated or copied directly by the cellular machinery. The first viral RdRp was discovered in the early 1960’s from poliovirus (PV). The poliovirus RNA polymerase (PV3D) is one of the best-studied viral RdRp and is often used as a reference for other newly identified RdRps. Studies including structural, RNA binding, nucleoside triphosphate (NTP) binding, polymerization of nucleotides, RNA strand displacement, and interactions with other viral proteins have been thoroughly investigated for PV3D [75, 76, 77, 78].
Most of our understanding on the properties of viral RdRps comes from
The RdRp gene of noroviruses is located at the C-terminal of non-structural polyprotein. With an approximate size of 57.5 kDa (in MNV), this virally encoded non-structural (NS7 in MNV) protein plays a key role in norovirus G RNA and SG RNA replication. Generally, the replication of G RNA is achieved through a negative sense RNA intermediate which serves as a template for the production of nascent positive sense viral G RNA. This general mechanism also applies to the caliciviruses where the presence of negative sense G RNA as well as SG RNA has been shown by Northern blot analysis during the infection of FCV in tissue culture. Currently, four main mechanisms for the initiation of RNA synthesis by recombinant calicivirus (including norovirus) RdRps have been demonstrated
5. Production of subgenomic RNA in other viruses relative to norovirus
The genome organization and strategies for gene expression of positive strand RNA viruses are diverse. In addition to the occurrence of specific proteolytic cleavage sites which mediate the translational processing of the large polyprotein and give rise to several mature proteins encoded by one large ORF, many viruses often express their downstream ORFs through the transcription and translation of a SG RNA. Generally, SG RNAs of positive strand RNA viruses are identical to the 3′ ends of their parental G RNA. However, they vary in length where the 5′ end of these SG RNAs are in proximity with the start codon of respective ORF. In most cases, these viral SG RNAs carry the ORFs that code for proteins required in the intermediate and late stages of infection, such as the structural proteins. Animal positive stranded RNA viruses that produce SG RNA include the
There are currently two well-characterized and one additional mechanism for positive strand RNA virus SG RNA synthesis. The first described mechanism and the most widely recognized model is internal initiation, which has been clearly demonstrated in studies involving brome mosaic virus (BMV) (
Animal viruses such as Sindbis virus (alphavirus) and Rubella virus (rubivirus) from the
The second mechanism for SG RNA synthesis is termed as a premature termination and occurs during the (−) strand template synthesis from the full length (+) strand G RNA. This premature termination gives rise to a subgenomic-length (−) strand RNA that then serves as a template for subsequent end-to-end (+) strand SG RNA synthesis. The generation of this smaller subgenomic-length (−) strand complementary RNA is due to the early disengagement of the RdRp when it reaches a RNA secondary structure in the (+) strand viral genome template (known as a termination signal). These RNA structures are normally comprised of either local secondary structures or long-distance RNA interactions that form a highly ordered structure. The plant virus tomato bushy stunt virus (TBSV), the prototype member of the
In addition to the two well-characterized SG RNA synthesis mechanisms described above, there is another more unusual mechanism employed by members of the families
6. The norovirus subgenomic RNA transcript and its translational products
All noroviruses produce a SG RNA during their replication cycle in infected cells. This SG RNA is 3′ co-terminal with the full-length G RNA, has VPg linked at the 5′ end and carries a poly-A tail at the 3′ end. Typically, the SG RNAs of noroviruses contains ORF2 and ORF3 (and ORF4 in the case of MNV and some sapoviruses) which code for viral structural proteins (VP1 and VP2). The production of a SG RNA message may act to delay the production of structural proteins until the initial rounds of viral replication have taken place. Both positive and negative sense SG RNA intermediates (~2.5 kb in length) can be detected by northern blot analysis of purified FCV replication complexes .
Following the transcription of MNV SG RNA, the expression of this messenger transcript via VPg-dependent translation initiation is achieved as described for the G RNA. The 5′ proximal ORF2, which encodes the major capsid protein is first translated when the scanning ribosomal complex encounters the first AUG codon, a typical strategy for translation. However, in viruses with polycistronic SG RNAs, the translation of their 3′ terminal ORF is not as efficient as the preceding ORF. Therefore, many viruses employ several strategies to provide sufficient access for ribosomes to downstream ORFs. These strategies include leaky scanning of 40S subunits past the start codon of the first ORF, the possession of intercistronic internal ribosome entry signal, programmed ribosomal frame-shifting during elongation and stop codon suppression at the termination step . All noroviruses SG RNA are bicistronic messages. The translation of the 3′ proximal ORF in this case is achieved by a unique mechanism called termination reinitiation. In this mechanism, a proportion of the 40S ribosomal subunit remains associated with the mRNA following the translational termination at the preceding stop codon. This enables reinitiation at the AUG of a downstream ORF, which is in close proximity. This characteristic has been observed for different caliciviruses where the initiation codon of VP2 (overlapped with VP1) is only 2 nucleotides away from the stop codon of VP1 for RHDV. Meanwhile for Norwalk virus, FCV and MNV, the start codon of VP2 is overlapped with the stop codon of VP1 . Other than the close proximity between the stop and start codon, the efficiency of termination-reinitiation translation is also determined by a stretch of 70 to 80 nucleotides upstream of the stop-start window which facilitates the transit of the ribosome through the stop codon of VP1. This region of conserved sequence is termed TURBS (termination upstream ribosome binding site motif). The translation of VP2 from the FCV, RHDV and MNV SG RNA is dependent on this TURBS region, which is located immediately upstream of the VP1 stop codon [110, 111, 112]. The TURBS contain two important sequences; the 5′ sequence (termed as Motif 1) is proposed to function in binding the 18S rRNA (through complementary sequence) whilst the other sequence is thought to be important in tethering the ribosome to enable translation of VP2 at the correct site [110, 113]. Alternatively, the TURBS may also act by interacting with eIF3 or eIF3/40S ribosome complexes preventing disassembly of the ribosome following VP1 translation termination. This alternative mechanism is supported by the fact that purified eIF3 is able to stimulate translational re-initiation .
7. The replication of norovirus subgenomic RNA
The presence of SG RNA of norovirus in infected cells is often used as indicator for active viral genome replication. Importantly, the mechanism that is used by noroviruses to achieve their SG RNA transcription is poorly understood until very relatively recently. Initial evidence from
As reported by Simmonds et al. , a mutant cDNA clone containing a series of non-coding mutations called m53 that destabilized the RNA structure was generated. These mutations were designed to destabilize the stem loop structure by weakening the base pairing without affecting the NS7 coding sequence. This mutated cDNA clone was used in the DNA-based reverse genetics system and reported to cause a lethal phenotype effect, whereby no infectious virus can be detected by TCID50 in the recoveries. However, by compensating the initial m53 mutations to restore the base pairing within the stem loop structure (called m53r), a viable virus was recovered with a titer close to that of the wild type virus. This series of experiments concluded that RNA stem loop structure is important for viral replication and might function as part of the SG RNA promoter. Even though the m53 mutation disrupting SLa5045 caused a lethal phenotype, serial “blind” passage of the recoveries (from the DNA based reverse genetics system) in RAW264.7 cells often produced viable viruses. Sequence analysis revealed that these viable viruses contained two types or classes of mutation. The first class were phenotypic-revertant viruses where nucleotide changes were identified that resulted in partial reformation of the SLa5045 stem loop structure. The ability to isolate phenotypic revertant viruses that repaired the defective RNA structure was not unexpected as the stem loop structure is predicted to play an important role in viral replication. This observation indicates that m53 mutation in the viral genome results in poor viral genome replication in tissue culture. Phenotypic-revertant mutations arise in tissue culture and those that promote replication are favored and amplified during the serial “blind” passage until they become dominant. Another type of mutation observed were suppressor mutations, whereby the m53 mutation in the SLa5045 was still present, but changes outside the stem loop structure, within the NS7 coding region, were also identified . Further characterization of this suppressor mutant viruses in cell culture revealed that they possess a slower growth kinetics, lower-level proteins production and lower-level of G RNA and SG RNA transcripts synthesis compared to WT virus . More importantly, these data indicate that nucleotide changes were responsible for the suppression phenotype rather than any amino acid change, suggesting the potential involvement of long-range RNA–RNA interactions between SLa5045 and a region ~100 nucleotides upstream of this Sla5045 stem loop structure . However, this hypothesis is yet to be proven with scientific experimental and the current readily available bioinformatics tools are not adequate to accurately predict such long range RNA–RNA interaction. On the other note, such long range RNA–RNA interactions between promoter regions are well established with some sequences being up to ~1500 nucleotides apart e.g. the nodavirus Flock House virus (FHV) and tombusvirus tomato bushy stunt virus (TBSV) have been documented to contain such interactions even though these viruses employ a premature termination mechanism for their SG RNA synthesis [118, 119]. In the case of MNV however, it is worth to note that this long-range RNA–RNA interaction presumably occurs on the negative strand RNA to produce a suppression effect on m53 mutation of the SLa5045.
Utilizing the MNV reverse genetics system, virus recoveries using series of modified cDNA with additional copy of SLa5045 in
Even though the presence of low levels of negative sense SG RNA have been argued for the premature termination of negative sense G RNA during elongation by RdRp that produces SG-length negative sense RNA transcript (act as template for positive sense SG RNA) [73, 120], a more detailed study suggests that norovirus SG RNA replication follows the internal initiation mechanism. Employing genetics and biochemical tools, a recent study demonstrates that accurate norovirus SG RNA synthesis is depend on a sequence and genotype-specific interaction of the viral RdRp with a stem-loop sequence (SLa5045) on the minus-strand RNA . In that study, the investigators performed an
The synthesis of norovirus SG RNA is a clear signal for the existence of genome replication since the production of this smaller RNA (that is 3′ co-terminal with the full length viral genome) is dependent on efficient genome replication in infected cells. Furthermore, the transcription of SG RNA at the middle and latter stages of infection is also thought to regulate the production of infectious virions. Since the capsid proteins of noroviruses are translated from the SG RNA messenger, the encapsidation process is initiated once the viral RNA replication begins. Investigations on the involvement of functional RNA elements in regulating the synthesis of the MNV SG RNA were carried out extensively to determine the mechanism employed by noroviruses in their genome replication accurately. Based on the establised data available recently, now clear that we could confidently presume that norovirus follows the internal initiation mechanism for the synthesis of SG RNA. The studies also proved the crucial role of small stem loop/hairpin structure within the coding region of NS7 in the viral replication.
The author would like to acknowledge Research University Grant (RUI), Universiti Sains Malaysia (1001/CIPPT/8012205).
Conflict of interest
The author declares no conflict of interest.