Positivesense Rna Viruses Encoding More Than One Translational Reading Frame

A positive-sense RNA virus that must regulate gene expression while infecting a eukaryotic host faces a fundamental problem: The eukaryotic ribosome cannot initiate translation of an ORF following translation of one upstream of it. While a positive-sense RNA virus genome could (and some do) contain more than one ORF, these ORFs cannot be independently translated at different rates during infection without some means to overcome this fundamental mechanistic limitation.

One way to overcome the problem is for a virus to encapsidate more than one mRNA (in other words, for the virus to contain a segmented genome). This approach is utilized by a number of positive-sense RNA viruses infecting plants, but has not been described for animal viruses. This finding is somewhat surprising since there are numerous negative-sense RNA viruses with segmented genomes that are successful animal and human pathogens. The list contains influenza viruses, hantaviruses, and arenaviruses.

Despite the disinclination of positive-sense RNA viruses that infect animal cells to encapsid-ate segmented genomes, another strategy for regulating mRNA expression is utilized successfully. This strategy involves the encoding of a cryptic (hidden) ORF in the genomic RNA, which can be translated from a viral mRNA generated by a transcription step during the replication cycle. With this strategy, viral gene expression from the full-length positive-sense mRNA contained in the virion results in translation of a 5' ORF, and this protein (an enzyme) is involved in generation of a second, smaller mRNA by transcription.

The second mRNA (which is not found in the virion), in turn, is translated into a distinct viral protein. Such a scheme allows the nonstructural proteins encoded by the virus — the enzymes required for replication — to be expressed in lesser amounts or at different times in the infection cycle than the proteins ending up in the mature virion. Clearly, this approach is effective as witnessed by the number of important pathogens that utilize it.

Two viral mRNAs are produced in different amounts during togavirus infections

Togaviruses are enveloped RNA viruses that display a complex pattern of gene expression during replication. Sindbis virus is a well-studied example. This arthropod-borne virus causes only very mild diseases in (rare) humans, but its size and relative ease of manipulation make it a useful laboratory model for the group as a whole.

Sindbis virus has a capsid structure similar to picornaviruses and flaviviruses, and like flavi-viruses, the capsid is enveloped. The viral genome contains two translational ORFs. Initially, only the first frame is translated into viral replication enzymes. These enzymes both replicate the virion RNA and generate a second mRNA that encodes viral structural proteins.

The viral genome

Sindbis virus and its 11,700-base genome is shown in Fig. 14.6. The virion genomic RNA (termed 49s RNA for its sedimentation rate in rate zonal centrifugation - see Chapter 11, Part III) has a capped 5' end and a polyadenylated 3' end. Both capping and polyadenylation appear to be carried out by viral replication enzymes, possibly in a manner somewhat analogous to that seen for the negative-sense vesicular stomatitis virus (VSV), which is discussed in Chapter 16.

The Sindbis virus genome contains two ORFs. The 5' ORF encodes a replication protein precursor that is processed by proteases to generate four different replicase polypeptides. The 3' ORF encodes capsid protein and envelope glycoproteins.

The virus replication cycle

Virus entry Viral entry is via receptor-mediated endocytosis as shown in Fig. 14.7(a). The entire virion, including envelope, is taken up in the endocytotic vesicle. Acidification of this vesicle leads to modification of the viral membrane glycoprotein. This allows the viral

Fig. 14.6 Sindbis virus — a typical togavirus. The virion (6070 nm in diameter) and genetic map are shown. The Sindbis genome contains two translational reading frames; only the upstream (5') one can be translated from the approximately 11,000-nucleotide (nt) capped and polyadenylated 49s (positive) virion-associated genomic RNA. This upstream translational frame encodes nonstructural proteins via expression of two precursor proteins. The larger, which contains the polymerase precursor, is translated by suppression of an internal stop codon in the reading frame.

membrane to fuse with the vesicle, and causes the capsid to disrupt so that viral genomic mRNA is released into the cytoplasm.

Early gene expression As shown in Fig. 14.7(b), only the 5' ORF can be translated from intact viral mRNA, because the eukaryotic ribosome falls off the viral mRNA when it encounters the first translation stop signal (either UAA, UAG, or UGA - see Chapter 13). With Sindbis virus, this situation is complicated by the fact that this first ORF in the genomic RNA contains a stop signal about three-quarters of the way downstream of the initiation codon. This termination codon can be recognized to generate a shorter precursor to the nonstructural proteins, but it can also be suppressed. (In genetics, the term suppression refers to the cell periodically ignoring a translation stop signal either because of an altered tRNA or a ribosomal response to secondary structure in the mRNA encoding it.) With Sindbis virus infection, the suppression is ribosomal, and results in about 25% of the nonstructural precursor protein containing the remaining information shown in ORF-1 in the genetic map. As discussed in Chapter 19, suppression of an internal stop codon also has a role in the generation of retrovirus protein.

In Sindbis virus infection, translation of infectious viral RNA generates replication enzymes that are derived by autoproteolytic cleavage (i.e., self-cleavage) of the replicase precursor protein. This can be considered an "early" phase of gene expression; however, things happen fast in the infected cell and this may only last for a few minutes.

Viral genome replication and generation of 26s mRNA The replication enzymes expressed from genomic 49s positive-sense mRNA associated with genomic RNA to generate 49s negative-sense RNA through RI-1 are shown in Fig. 14.8(a). The next step in the process is critical to regulated expression of the two virus-encoded precursor proteins. With Sindbis, the negative-sense RNA complementary to genomic positive-sense RNA is the template for two different positive-sense mRNAs. Both are capped and polyadenylated. The first is more 49s positive-sense virion RNA. The second is 26s positive-sense RNA. The shorter 26s mRNA is generated by replicase beginning transcription of negative-sense RNA in the middle and generating a "truncated" or

Sindbis virus

Sindbis virus

Replicase genes | Structural genes

Suppressible stop codon

Sindbis virion

Viral Receptor Sindbis
(b)

49s genomic RNA (positive strand) ORF-1 (~7000 nt)

Suppressible Translation of stop codon nonstructural genes

Cleavages

NSP1 NSP2

(540aa) (807aa) Protease, helicase

NSPS NSP4 (549aa) (610aa) Pol

(reduced molar amount of Pol expressed in relation to other replication proteins)

Fig. 14.7 The early stages of Sindbis virus infection. (a) The first step is receptor-mediated endocytosis, leading to fusion of the viral membrane with that of the endocytotic vesicle, which leads to release of the Sindbis virus genome (mRNA) into the infected cell's cytoplasm. As outlined in Chapter 6, Part II, internalization of the enveloped virion within an endocytotic vesicle is followed by acidification and covalent changes in membrane proteins. This results in fusion of the viral membrane with that of the endocytotic vesicle and release of the viral genome. (b) Translation of the virion RNA results in expression of the precursors to the nonstructural replicase and other viral proteins encoded in the 5' translational reading frame. These proteins mediate replicase, capping, and protease functions.

Fig. 14.8 (a) The replication of Sindbis virus genome, and generation of the subgenomic 26s mRNA. This mRNA is expressed by an internal start site for viral replicase, and is translated into structural proteins since it encodes only the open reading frame (ORF) that was cryptic in the 49s positive-sense virion RNA. (b) Sindbis virus structural proteins are translated as a single precursor. When the N-terminal capsid protein is cleaved from the precursor, a signal sequence consisting of a stretch of aliphatic amino acids associates with the endoplasmic reticulum. This association allows the membrane protein portion of the precursor to insert into the lumen of the endoplasmic reticulum. As the protein continues to be inserted into the lumen, it is cleaved into smaller product proteins by cellular enzymes. Cellular enzymes also carry out glycosylation.

Suppressible Replicase enzymes stop codon encoded by ORF-1

RI-1

Interior replicase start

Replicase

Interior replicase start

RI-2

Replicase

Fig. 14.8 (a) The replication of Sindbis virus genome, and generation of the subgenomic 26s mRNA. This mRNA is expressed by an internal start site for viral replicase, and is translated into structural proteins since it encodes only the open reading frame (ORF) that was cryptic in the 49s positive-sense virion RNA. (b) Sindbis virus structural proteins are translated as a single precursor. When the N-terminal capsid protein is cleaved from the precursor, a signal sequence consisting of a stretch of aliphatic amino acids associates with the endoplasmic reticulum. This association allows the membrane protein portion of the precursor to insert into the lumen of the endoplasmic reticulum. As the protein continues to be inserted into the lumen, it is cleaved into smaller product proteins by cellular enzymes. Cellular enzymes also carry out glycosylation.

RI-2

AAA

subgenomic mRNA. The region on the negative-sense strand where the transcriptase binds is roughly analogous to a promoter, but its sequence does not exhibit the features of promoters found in DNA genomes.

Generation of structural proteins The short 26s mRNA contains only the second ORF contained in the full-length genomic RNA. This ORF was hidden or inaccessible to translation of the full-length virion mRNA. With the 26s mRNA, however, cellular ribosomes can translate the ORF into precursors of capsid and envelope proteins. Expression of structural proteins, thus, requires at least partial genome replication and is generally termed late gene expression, although it occurs very soon after infection. Translation of the 5' region of late 26s mRNA generates capsid protein that is cleaved from the growing peptide chain by proteolytic cleavage. This cleavage generates a new N-terminal region of the peptide. The new N-terminal region of the peptide contains a stretch of aliphatic amino acids, and the hydrophobic nature of this "signal' sequence results in the growing peptide chain inserting itself into the endoplasmic reticulum in a manner analogous to synthesis of any cellular membrane protein. This process is shown in Fig. 14.8(b).

Mab Glycosylation Mechanism Golgi Body

Fig. 14.8 Continued

Following initial insertion of the membrane proteins' precursor, the various mature proteins are formed by cleavage of the growing chain within the lumen of the endoplasmic reticulum. This maturational cleavage is carried out by cellular proteins.

Posttranslational processing, such as glycosylation of membrane-associated components of the late structural protein, takes place in the Golgi apparatus, and viral envelope protein migrates to the cell surface. Meanwhile, capsid formation takes place in the cytoplasm, genomes are added, and the virion is formed by budding through the cell surface, as described in Chapter 6, Part II.

Togavirus cytopathology and disease

The replication process of togaviruses is a step more complex than that seen with picornaviruses, and the cell needs to maintain its structure to allow continual budding of new virus. Accordingly, there is less profound shutoff of host cell function until a long time after infection. A major cytopathic change is alteration of the cell surface. This can lead to fusion with neighboring cells so that virus can spread without ever leaving the first infected cells. This alteration to the cell surface also involves antigenic alteration of the cell. Such types of cytopa-thology are found with many enveloped RNA viruses, whether they are positive or negative sense.

Based upon the number of viruses identified as belonging to the group, the togaviruses are an extremely successful group of viruses, and like the flaviviruses, many are transmitted by arthropods. As noted in Chapter 5, Part II, it is for this reason that these two groups of positive-sense RNA viruses are termed arboviruses (arthropod-borne viruses). While this terminology is convenient for some purposes, it does not recognize significant differences in the replication strategies of these two groups of viruses. Further, numerous other types of viruses are spread by arthropod vectors, and some togaviruses and flaviviruses are not transmitted by such vectors. A striking example is rubella (German measles) virus.

Many togaviruses cause sporadic outbreaks of mosquito-borne encephalitis because they have a propensity for replication in cells making up the brain's protective lining. Although such disease can be severe, many forms have a favorable prognosis with proper medical care, as neurons are not the primary targets of infection.

The only known host for rubella virus is humans. The virus causes generally mild and often asymptomatic diseases in children and adults, although a mild rash may be evident. Despite the generally benign course of infection, it is remarkable that rubella is associated with a diverse group of clinical diseases, including rubella arthritis and neurological complications.

Periodic local epidemics are characteristic of rubella virus infections, and although the virus induces an effective immune response, the endemic nature of the virus ensures that once a large-enough pool of susceptible individuals arises, sporadic regional epidemics occur. The major problem with these periodic occurrences is the very fact that the disease is often so mild as to be asymptomatic in adults of childbearing age. While the symptoms are very mild for adults and children, this is not the case for fetal infections. Infection of the mother in the first trimester of pregnancy often leads to miscarriage, and a fetus who survives is almost inevitably severely developmentally impaired. Infection of the mother later in pregnancy has a more benign outcome.

The tragedy of rubella infections is that although there are effective vaccines, the disease is often so mild that an individual can be infected and can spread the virus without knowing it. For this reason, women of childbearing age who are in contact with young children or other adults at risk of infection should be vaccinated.

A somewhat more complex scenario of multiple translational reading frames and subgenomic mRNA expression: coronavirus replication

Even more complex scenarios exist for expression and regulation of gene function in infections by positive-sense RNA viruses. The replication strategy of the coronaviruses is a good example of such complexity. Coronaviruses and toroviruses are members of the Coronaviridae and, together with the Arteriviridae and Roniviridae, make up the larger grouping called the order Nidovirales (nido = nested). The structure of coronaviruses is shown in Fig. 14.9 — the helical nucleocapsid is unusual for a positive-sense RNA virus.

The nucleocapsid is helical within a roughly spherical membrane envelope, and the envelope glycoproteins project as distinct "spikes" from this envelope. These glycoprotein spikes from the lipid bilayer appear as a distinctive crown-like structure in the electron microscope, hence, the name corona (crown)-viruses.

The 30-kb coronavirus genome encodes at least five separate translational reading frames, and is the template for the synthesis of at least six subgenomic mRNAs. Each subgenomic mRNA

Coronavirus

30,000 nt

Coronavirus

Lipid bilayer

Nonstructural

NS E1

Lipid bilayer

Nonstructural

NS E1

i i 200K polymerase 1 -

lili i-1 14K NS

lili i i 14K NS

lili

Common leader

AAAA 3'

AAAA 3'

AAAA 3'

AAAA 3'

AAAA 3'

AAAA 3'

AAAA 3'

AAAA 3'

Common 3' end

Fig. 14.9 A schematic representation of the coronavirus virion. This is the only known group of positive-sense RNA viruses with a helical nucleocapsid. The name of the virus is derived from appearance of the glycoproteins projecting from the envelope, which gives the virus a crown-like shape. The diameter of the spherical enveloped virion ranges between 80 and 120 nm depending on experimental conditions in visualization. The 30,000-nucleotide (nt) capped and polyadenylated positive-sense genome encodes five translational reading frames that are expressed through translation of the genomic RNA and six subgenomic positivesense mRNAs. These capped and polyadenylated subgenomic mRNAs each have the same short 5' leader and share nested 3' sequences. Although two models exist for the production of this nested set, the most likely at this time appears to be that they are derived by transcription of subgenomic negative-sense templates, produced by discontinuous copying of the viral genomic RNA.

contains a short, identical leader segment at the 5' end that is encoded within the 5' end of the genomic RNA. All subgenomic mRNAs have the same 3' end, and thus are a nested set of transcripts, giving the name to the order Nidovirales. Only the 5' translational reading frame is recognized in each, and the others are cryptic. These features are also shown in Fig. 14.9.

Coronavirus replication

Coronavirus replication involves the generation and translation of genomic and subgenomic viral mRNAs as shown in Fig. 14.10. Virus entry is by receptor-mediated fusion of the virion with the plasma membrane followed by release of genomic RNA. A good deal of recent work concerning viral replication has been stimulated by the identification of a coronavirus as

Coronavirus Replication Review
  1. 14.10 The replication cycle of a coronavirus. Replication is entirely cytoplasmic. Infection is initiated by receptor-mediated membrane fusion to release the genomic mRNA. This RNA is translated into the very large (>200 kd) polymerase/capping enzyme. The interaction between full-length virion positive-sense RNA and replicase generates the templates for the mRNAs. Two models are proposed for the synthesis of subgenomic mRNA: leader-primed synthesis and discontinuous negative-strand synthesis. The second of these two models is shown in the figure. The result of both models is the synthesis of a nested set of mRNAs that contain the same 5' leader sequence and overlapping 3' ends. Translation of the various subgenomic mRNAs leads to synthesis of the various structural and nonstructural proteins encoded by interior translational reading frames. The mature virions assemble and become enveloped by budding into intracytoplasmic vesicles; these exocytotic vesicles then migrate to the cell surface where virus is released. At later times, cell lysis occurs.
  2. 14.10 The replication cycle of a coronavirus. Replication is entirely cytoplasmic. Infection is initiated by receptor-mediated membrane fusion to release the genomic mRNA. This RNA is translated into the very large (>200 kd) polymerase/capping enzyme. The interaction between full-length virion positive-sense RNA and replicase generates the templates for the mRNAs. Two models are proposed for the synthesis of subgenomic mRNA: leader-primed synthesis and discontinuous negative-strand synthesis. The second of these two models is shown in the figure. The result of both models is the synthesis of a nested set of mRNAs that contain the same 5' leader sequence and overlapping 3' ends. Translation of the various subgenomic mRNAs leads to synthesis of the various structural and nonstructural proteins encoded by interior translational reading frames. The mature virions assemble and become enveloped by budding into intracytoplasmic vesicles; these exocytotic vesicles then migrate to the cell surface where virus is released. At later times, cell lysis occurs.

the agent of the emerging disease SARS (discussed in more detail below and in Chapter 25, Part V).

Virus entry is by receptor-mediated fusion of the virion with the plasma membrane followed by release of genomic RNA. The receptor for the SARS coronavirus is angiotensin converting enzyme 2 (ACE2). The importance of this virus—receptor interaction will be discussed below with respect to the pathogenesis of the SARS agent.

This RNA (one of the largest mRNAs characterized) is translated into a replication protein that, interestingly, is encoded in an ORF encompassing 70% of the virus's coding capacity. The reason why coronavirus replication proteins are encoded by such a large gene is not yet known.

The mature replication proteins derived from the first translation product are used to produce all subsequent mRNA species. There are two competing models that have been presented for coronavirus transcription (Fig. 14.10): leader-primed transcription and discontinuous transcription during negative-strand synthesis.

Leader-primed transcription proposes that the replications proteins first produce a full-length negative strand copy of the genome, using a standard RI-1 structure. From this template is then transcribed multiple copies of the extreme 3' end, called the leader region. These leader transcripts then function to prime synthesis of subgenomic mRNAs, initiated at homologous regions in between each of the genes (intergenic sequences).

Discontinuous transcription during negative-strand synthesis proposes that the replication proteins transcribe negative-strand copies of the genome, using RI-1 structures. Some of these products are subgenomic. These subgenomic species are produced when the replicase complex in the RI-1 pauses at the intergenic regions and then jumps to the end of the genome, copying the leader sequence. The result of this step is a subgenomic negative strand RNA that is the complement of the mRNA. Subsequent transcription of this template produces the mRNA itself, using RI-2 structures that are also subgenomic.

Evidence can be obtained in support of both of these models and both result in mRNAs that have common 5' sequences (the leader) and common 3' regions. This nested set of mRNAs is observed during coronavirus infection. Both full-length and subgenomic replicative intermediates can be found in cells at various times after infection. At this writing, much of the evidence obtained with the SARS coronavirus and with other related viruses tends to support the second of these models, that is, discontinuous transcription during negative strand synthesis.

The specific mechanism of the transcriptase jumping in each model is proposed to involve transcriptional regulating sequences that contain core elements recognized in protein—RNA interactions. The net result, however, is that each mRNA has the same 5' leader sequence and therefore has only has one sequence of RNA needing to be capped. The addition of the polyA tracts onto the individual mRNAs also only requires the recognition of one sequence on the positive-sense template by viral replicase, since all mRNAs have the same 3' end. An alternative possibility is that the polyA is template-derived, coming from transcription of a common polyU sequence present at the 5' end of the subgenomic negative strands.

Cytopathology and disease caused by coronaviruses

Certain coronaviruses, along with the rhinoviruses, can cause mild and localized respiratory tract infections (head colds). The mildness of colds results from a number of both viral and cellular factors. First, the viruses causing the common cold have a very defined tissue tropism for nasopharynx epithelium. Spread of the virus is limited by ill-defined localized immune factors of the host. The ability of a cold virus infection to remain localized at the site of initial infection is a great advantage to the virus. Local irritation leads to sneezing, coughing, and runny nose — all important for viral spread. Mildness and localization of the infection tend to limit the immune response, which is another distinct advantage. A mild infection results in short-lived immunity, and this, along with the fact that a large number of serotypes exist as a result of the high error frequency of the genome replication process, mean that colds are a common and constant affliction.

In the late winter and spring of 2003 a new illness broke out, focused in China and Singapore. Severe acute respiratory syndrome (SARS) proved to be more than the common cold, having a case fatality rate of 10—20%. The etiologic agent of SARS is a coronavirus, named SARS-CoV. Although the original transmission to humans was apparently from the civet cat, a recent report suggests that the natural reservoir host for SARS-CoV is one of several species of bats.

SARS-CoV has been shown to utilize the cellular protein ACE2 (angiotensin-converting enzyme 2) as a receptor to initiate viral entry into the cytoplasm of the infected cell. One model that has been proposed for the high mortality induced by this virus involves the role of angiotensin in acute lung injury. ACE2 converts angiotensin from a form that induces tissue damage and lung edema into a form of the protein that is more benign. Infection with SARS-CoV appears to cause downregulation of this enzyme, an event that is proposed to be significant in the pathogenesis of this virus.

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