Basic characteristics of a virus viral replication




















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In this case, the core still contains dozens of proteins and at least 10 distinct enzymes. The structure and chemistry of the nucleocapsid determines the subsequent steps in replication.

Reverse transcription can only occur inside an ordered retrovirus core particle and does not proceed to completion with the virus RNA free in solution. Eukaryotic viruses which replicate in the nucleus, such as members of the Herpesviridae , Adenoviridae , and Polyomaviridae , undergo structural changes following penetration, but overall remain largely intact.

This is important because these capsids contain nuclear localization sequences responsible for attachment to the cytoskeleton and this interaction allows the transport of the entire capsid to the nucleus. At the nuclear pores, complete uncoating occurs and the nucleocapsid passes into the nucleus. The replication strategy of a virus depends, in large part, on the structure and composition of its genome.

For viruses with RNA genomes in particular, genome replication and transcription are often inextricably linked, and frequently carried out by the same enzymes. Therefore, it makes most sense to consider both of these aspects of virus replication together. Replication is exclusively nuclear or associated with the nucleoid of prokaryotes. The replication of these viruses is relatively dependent on cellular factors. In some cases, no virus-encoded enzymes are packaged within these virus particles as this is not necessary, whereas in more complex viruses numerous enzymatic activities may be present within the particles.

Replication occurs in cytoplasm. These viruses have evolved or acquired from their hosts all the necessary factors for transcription and replication of their genomes and are therefore largely independent of the cellular apparatus for DNA replication and transcription. Because of this independence from cellular functions, these viruses have some of the largest and most complex particles known, containing many different enzymes. The replication of these virus genomes occurs in the nucleus, involving the formation of a double-stranded intermediate which serves as a template for the synthesis of new single-stranded genomes.

In general, no virus-encoded enzymes are packaged within the virus particle since most of the functions necessary for replication are provided by the host cell. These viruses all have segmented genomes, as each segment is transcribed separately to produce individual monocistronic messenger RNAs. Replication occurs in the cytoplasm and is largely independent of cellular machinery, as the particles contain many virus-encoded enzymes essential for RNA replication and transcription since these processes involving copying RNA to make further RNA molecules do not normally occur in cellular organisms.

Viruses with polycistronic mRNA such as flaviviruses and picornaviruses. As with all the viruses in this group, the genome RNA represents mRNA which is translated after infection, resulting in the synthesis of a polyprotein product, which is subsequently cleaved to form the mature proteins. Viruses with complex transcription such as coronaviruses and togaviruses. In this subgroup, two rounds of translation are required to produce subgenomic RNAs which serve as mRNAs in addition to the full-length RNA transcript which forms progeny virus genomes.

Although the replication of these viruses involves copying RNA from an RNA template, no virus-encoded enzymes are packaged within the genome since the ability to express genetic information directly from the genome without prior transcription allows the virus replicase to be synthesized after infection has occurred.

The genomes of these viruses can also be divided into two types. A complete replication cycle involves conversion of the RNA form of the virus genetic material into a DNA form, the provirus, which is integrated into the host cell chromatin. The enzyme reverse transcriptase needs to be packaged into virus particles to achieve this conversion, as virus genes are only expressed from the DNA provirus and not from the RNA genome found in retrovirus particles of retroviruses.

This group of viruses also relies on reverse transcription, but unlike the retroviruses, this occurs inside the virus particle during maturation. On infection of a new cell, the first event to occur is repair of the gapped genome, followed by transcription. As with group VI viruses, a reverse transcriptase enzyme activity is present inside virus particles, but in this case, the enzyme carries out the conversion of virus RNA into the DNA genome of the virus inside the virus particle.

This contrasts with retroviruses where reverse transcription occurs after the RNA genome has been released from the virus particle into the host cell. During assembly, the basic structure of the virus particle is formed as all the components necessary for the formation of the mature virion come together at a particular site in the cell.

The site of assembly depends on the pattern of virus replication and the mechanism by which the virus is eventually released from the cell and so varies for different viruses.

Although some DNA virus particles form in the nucleus, the cytoplasm is the most common site of particle assembly. In the majority of cases, cellular membranes are used to anchor virus proteins, and this initiates the process of assembly. For enveloped viruses, the lipid covering is acquired through a process known as budding, where the virus particle is extruded through a cell membrane.

Lipid rafts are membrane microdomains enriched in glycosphingolipids or glycolipids , cholesterol and a specific set of associated proteins. Lipid rafts have been implicated in a variety of cellular functions, such as apical sorting of proteins and signal transduction, but they are also used by viruses as platforms for cell entry e.

As with the earliest stages of replication, it is often not possible to identify the assembly, maturation, and release of virus particles as distinct and separate phases. The site of assembly has a profound influence on all these processes. In general terms, rising intracellular levels of virus proteins and genomes reach a critical concentration and this triggers assembly. Many viruses achieve high levels of newly synthesized structural components by concentrating these into subcellular compartments known as inclusion bodies.

These are a common feature of the late stages of infection of cells by many different viruses. Alternatively, local concentrations of virus structural components can be boosted by lateral interactions between membrane-associated proteins. This mechanism is particularly important in enveloped viruses which are released from the cell by budding see above. Maturation is the stage of the replication cycle at which virus particles become infectious. This often involves structural changes in the newly formed particle resulting from specific cleavages of virus proteins to form the mature products or from conformational changes in proteins which occur during assembly e.

Protein cleavage frequently leads to substantial structural changes in the capsid. Alternatively, internal structural alterations, for example, the condensation of nucleoproteins with the virus genome, often result in changes visible by electron microscopy.

Proteases are frequently involved in maturation, and virus-encoded enzymes, cellular proteases or a mixture of the two may be used.

Virus-encoded proteases are usually highly specific for particular amino acid sequences and structures, only cutting a particular peptide bond in a particular protein.

Moreover, they are often further controlled by being packaged into virus particles during assembly and only activated when brought into close contact with their target sequence by the conformation of the capsid, for example, by being placed in a local hydrophobic environment, or by changes of pH or cation cofactor concentrations inside the particle as it forms.

Retrovirus proteases are good examples of enzymes involved in maturation which are under tight control. The retrovirus core particle is composed of proteins from the gag gene and the protease is packaged into the core before its release from the cell on budding.

During the budding process, the protease cleaves the gag protein precursors into the mature products — the capsid, nucleocapsid, and matrix proteins of the mature virus particle. Other protease cleavage events involved in maturation are less closely controlled. Influenza A virus hemagglutinin must be cleaved into two fragments HA 1 and HA 2 to be able to promote membrane fusion during infection. Cellular trypsin-like enzymes are responsible for this process, which occurs in secretory vesicles as the virus buds into them prior to release at the cell surface; however, this process is controlled by the virus M2 protein, which regulates the pH of intracellular compartments in influenza virus-infected cells.

For lytic viruses most nonenveloped viruses , release is a simple process — the infected cell breaks open and releases the virus. The reasons for lysis of infected cells are not always clear, but virus-infected cells often disintegrate because viral replication disrupts normal cellular function, for example, the expression of essential genes.

Many viruses also encode proteins that stimulate or in some cases suppress apoptosis, which can also result in release of virus particles. Enveloped viruses acquire their lipid membrane as the virus buds out of the cell through the cell membrane, or into an intracellular vesicle prior to subsequent release.

Virion envelope proteins are picked up during this process as the virus particle is extruded. This process is known as budding. As mentioned earlier, assembly, maturation, and release are usually simultaneous processes for viruses which are released by budding.

The release of mature virus particles from their host cells by budding presents a problem in that these particles are designed to enter, rather than leave, cells.

Certain virus envelope proteins are involved in the release phase of replication as well as in receptor binding. Here we provide a brief introduction to coronaviruses discussing their replication and pathogenicity, and current prevention and treatment strategies.

Coronaviruses CoVs are the largest group of viruses belonging to the Nidovirales order, which includes Coronaviridae , Arteriviridae , Mesoniviridae , and Roniviridae families. The Coronavirinae comprise one of two subfamilies in the Coronaviridae family, with the other being the Torovirinae. The Coronavirinae are further subdivided into four genera, the alpha, beta, gamma, and delta coronaviruses. The viruses were initially sorted into these genera based on serology but are now divided by phylogenetic clustering.

All viruses in the Nidovirales order are enveloped, non-segmented positive-sense RNA viruses. They all contain very large genomes for RNA viruses, with some viruses having the largest identified RNA genomes, containing up to These differences cause significant alterations in the structure and morphology of the nucleocapsids and virions. The replicase gene encoding the non-structural proteins nsps occupies two-thirds of the genome, about 20 kb, as opposed to the structural and accessory proteins, which make up only about 10 kb of the viral genome.

Additionally, at the beginning of each structural or accessory gene are transcriptional regulatory sequences TRSs that are required for expression of each of these genes see Subheading 4. The accessory proteins are almost exclusively nonessential for replication in tissue culture; however, some have been shown to have important roles in viral pathogenesis [ 1 ]. An illustration of the MHV genome is depicted at the top.

Size of the genome and individual genes are approximated using the legend at the top of the diagram but are not drawn to scale. Coronavirus virions are spherical with diameters of approximately nm as depicted in recent studies by cryo-electron tomography and cryo-electron microscopy [ 2 , 3 ].

The most prominent feature of coronaviruses is the club-shaped spike projections emanating from the surface of the virion. These spikes are a defining feature of the virion and give them the appearance of a solar corona, prompting the name, coronaviruses. Within the envelope of the virion is the nucleocapsid. Coronaviruses have helically symmetrical nucleocapsids, which is uncommon among positive-sense RNA viruses, but far more common for negative-sense RNA viruses.

Coronavirus particles contain four main structural proteins. Homotrimers of the virus encoded S protein make up the distinctive spike structure on the surface of the virus [ 4 , 5 ]. The trimeric S glycoprotein is a class I fusion protein [ 6 ] and mediates attachment to the host receptor [ 7 ]. In most, coronaviruses, S is cleaved by a host cell furin-like protease into two separate polypeptides noted S1 and S2 [ 8 , 9 ].

S1 makes up the large receptor-binding domain of the S protein, while S2 forms the stalk of the spike molecule [ 10 ]. The M protein is the most abundant structural protein in the virion. It has a small N-terminal glycosylated ectodomain and a much larger C-terminal endodomain that extends 6—8 nm into the viral particle [ 12 ]. Despite being co-translationally inserted in the ER membrane, most M proteins do not contain a signal sequence.

Recent studies suggest the M protein exists as a dimer in the virion, and may adopt two different conformations, allowing it to promote membrane curvature as well as to bind to the nucleocapsid [ 13 ]. The coronavirus E proteins are highly divergent but have a common architecture [ 14 ].

The membrane topology of E protein is not completely resolved but most data suggest that it is a transmembrane protein. The E protein has an N-terminal ectodomain and a C-terminal endodomain and has ion channel activity.

As opposed to other structural proteins, recombinant viruses lacking the E protein are not always lethal, although this is virus type dependent [ 15 ]. The E protein facilitates assembly and release of the virus see Subheading 4. For instance, the ion channel activity in SARS-CoV E protein is not required for viral replication but is required for pathogenesis [ 16 ].

The N protein constitutes the only protein present in the nucleocapsid. It has been suggested that optimal RNA binding requires contributions from both domains [ 17 , 18 ].

N protein is also heavily phosphorylated [ 19 ], and phosphorylation has been suggested to trigger a structural change enhancing the affinity for viral versus nonviral RNA. N protein binds the viral genome in a beads-on-a-string type conformation. The genomic packaging signal has been found to bind specifically to the second, or C-terminal RNA binding domain [ 22 ]. N protein also binds nsp3 [ 18 , 23 ], a key component of the replicase complex, and the M protein [ 24 ].

These protein interactions likely help tether the viral genome to the replicase—transcriptase complex RTC , and subsequently package the encapsidated genome into viral particles. The protein acts as a hemagglutinin, binds sialic acids on surface glycoproteins, and contains acetyl-esterase activity [ 25 ].

These activities are thought to enhance S protein-mediated cell entry and virus spread through the mucosa [ 26 ]. Interestingly, HE enhances murine hepatitis virus MHV neurovirulence [ 27 ]; however, it is selected against in tissue culture for unknown reasons [ 28 ].

The initial attachment of the virion to the host cell is initiated by interactions between the S protein and its receptor. The S-protein—receptor interaction is the primary determinant for a coronavirus to infect a host species and also governs the tissue tropism of the virus.

Many coronaviruses utilize peptidases as their cellular receptor. It is unclear why peptidases are used, as entry occurs even in the absence of the enzymatic domain of these proteins. Following receptor binding, the virus must next gain access to the host cell cytosol.

This is generally accomplished by acid-dependent proteolytic cleavage of S protein by a cathepsin, TMPRRS2 or another protease, followed by fusion of the viral and cellular membranes. Fusion generally occurs within acidified endosomes, but some coronaviruses, such as MHV, can fuse at the plasma membrane. The formation of this bundle allows for the mixing of viral and cellular membranes, resulting in fusion and ultimately release of the viral genome into the cytoplasm.

The next step in the coronavirus lifecycle is the translation of the replicase gene from the virion genomic RNA. The replicase gene encodes two large ORFs, rep1a and rep1b, which express two co-terminal polyproteins, pp1a and pp1ab Fig. In most cases, the ribosome unwinds the pseudoknot structure, and continues translation until it encounters the rep1a stop codon.

Occasionally the pseudoknot blocks the ribosome from continuing elongation, causing it to pause on the slippery sequence, changing the reading frame by moving back one nucleotide, a -1 frameshift, before the ribosome is able to melt the pseudoknot structure and extend translation into rep1b, resulting in the translation of pp1ab [ 32 , 33 ]. It is unknown exactly why these viruses utilize frameshifting to control protein expression, but it is hypothesized to either control the precise ratio of rep1b and rep1a proteins or delay the production of rep1b products until the products of rep1a have created a suitable environment for RNA replication [ 34 ].

Polyproteins pp1a and pp1ab contain the nsps 1—11 and 1—16, respectively. In pp1ab, nsp11 from pp1a becomes nsp12 following extension of pp1a into pp1b. These polyproteins are subsequently cleaved into the individual nsps [ 35 ]. Coronaviruses encode either two or three proteases that cleave the replicase polyproteins.

They are the papain-like proteases PLpro , encoded within nsp3, and a serine type protease, the main protease, or Mpro, encoded by nsp5. Next, many of the nsps assemble into the replicase—transcriptase complex RTC to create an environment suitable for RNA synthesis, and ultimately are responsible for RNA replication and transcription of the sub-genomic RNAs. Interestingly, ribonucleases nspNendoU and nspExoN activities are unique to the Nidovirales order and are considered genetic markers for these viruses [ 37 ].

Viral RNA synthesis follows the translation and assembly of the viral replicase complexes. Sub-genomic RNAs serve as mRNAs for the structural and accessory genes which reside downstream of the replicase polyproteins.

Both genomic and sub-genomic RNAs are produced through negative-strand intermediates. Many cis-acting sequences are important for the replication of viral RNAs. Therefore, these different structures are proposed to regulate alternate stages of RNA synthesis, although exactly which stages are regulated and their precise mechanism of action are still unknown.

Perhaps the most novel aspect of coronavirus replication is how the leader and body TRS segments fuse during production of sub-genomic RNAs. This was originally thought to occur during positive-strand synthesis, but now it is largely believed to occur during the discontinuous extension of negative-strand RNA [ 48 ].

However, many questions remain to fully define the model. Answers to these questions and others will be necessary to gain a full perspective of how RNA replication occurs in coronaviruses. Finally, coronaviruses are also known for their ability to recombine using both homologous and nonhomologous recombination [ 50 , 51 ].

The ability of these viruses to recombine is tied to the strand switching ability of the RdRp. Following replication and sub-genomic RNA synthesis, the viral structural proteins, S, E, and M are translated and inserted into the endoplasmic reticulum ER. These proteins move along the secretory pathway into the endoplasmic reticulum—Golgi intermediate compartment ERGIC [ 52 , 53 ].

There, viral genomes encapsidated by N protein bud into membranes of the ERGIC containing viral structural proteins, forming mature virions [ 54 ]. The M protein directs most protein—protein interactions required for assembly of coronaviruses. However, M protein is not sufficient for virion formation, as virus-like particles VLPs cannot be formed by M protein expression alone.

When M protein is expressed along with E protein VLPs are formed, suggesting these two proteins function together to produce coronavirus envelopes [ 55 ]. The S protein is incorporated into virions at this step, but is not required for assembly. While the M protein is relatively abundant, the E protein is only present in small quantities in the virion.

Thus, it is likely that M protein interactions provide the impetus for envelope maturation. It is unknown how E protein assists M protein in assembly of the virion, and several possibilities have been suggested.

Some work has indicated a role for the E protein in inducing membrane curvature [ 57 — 59 ], although others have suggested that E protein prevents the aggregation of M protein [ 60 ].

The E protein may also have a separate role in promoting viral release by altering the host secretory pathway [ 61 ]. The M protein also binds to the nucleocapsid, and this interaction promotes the completion of virion assembly.

However, it is unclear exactly how the nucleocapsid complexed with virion RNA traffics to the ERGIC to interact with M protein and become incorporated into the viral envelope. Another outstanding question is how the N protein selectively packages only positive-sense full-length genomes among the many different RNA species produced during infection.

A packaging signal for MHV has been identified in the nsp15 coding sequence, but mutation of this signal does not appear to affect virus production, and a mechanism for how this packaging signal works has not been determined [ 22 ]. Furthermore, most coronaviruses do not contain similar sequences at this locus, indicating that packaging may be virus specific. Following assembly, virions are transported to the cell surface in vesicles and released by exocytosis.

It is not known if the virions use the traditional pathway for transport of large cargo from the Golgi or if the virus has diverted a separate, unique pathway for its own exit. In several coronaviruses, S protein that does not get assembled into virions transits to the cell surface where it mediates cell—cell fusion between infected cells and adjacent, uninfected cells.

This leads to the formation of giant, multinucleated cells, which allows the virus to spread within an infected organism without being detected or neutralized by virus-specific antibodies. Coronaviruses cause a large variety of diseases in animals, and their ability to cause severe disease in livestock and companion animals such as pigs, cows, chickens, dogs, and cats led to significant research on these viruses in the last half of the twentieth century. PEDV recently emerged in North America for the first time, causing significant losses of young piglets.

Porcine hemagglutinating encephalomyelitis virus PHEV mostly leads to enteric infection but has the ability to infect the nervous system, causing encephalitis, vomiting, and wasting in pigs. Feline enteric coronavirus FCoV causes a mild or asymptomatic infection in domestic cats, but during persistent infection, mutation transforms the virus into a highly virulent strain of FCoV, Feline Infectious Peritonitis Virus FIPV , that leads to development of a lethal disease called feline infectious peritonitis FIP.

FIP has wet and dry forms, with similarities to the human disease, sarcoidosis. However, additional research is needed to confirm this hypothesis. Bovine CoV causes significant losses in the cattle industry and also has spread to infect a variety of ruminants, including elk, deer, and camels. Infection of the reproductive tract with IBV significantly diminishes egg production, causing substantial losses in the egg-production industry each year [ 63 ].

More recently, a novel coronavirus named SW1 has been identified in a deceased Beluga whale [ 64 ]. Large numbers of virus particles were identified in the liver of the deceased whale with respiratory disease and acute liver failure. Although, electron microscopic images were not sufficient to identify the virus as a coronavirus, sequencing of the liver tissue clearly identified the virus as a coronavirus.

Finally, another novel family of nidoviruses, Mesoniviridae , has been recently identified as the first nidoviruses to exclusively infect insect hosts [ 66 , 67 ]. These viruses are highly divergent from other nidoviruses but are most closely related to the roniviruses. Interestingly, these viruses do not encode for an endoribonuclease, which is present in all other nidoviruses.

These attributes suggest these viruses are the prototype of a new nidovirus family and may be a missing link in the transition from small to large nidoviruses. The most heavily studied animal coronavirus is murine hepatitis virus MHV , which causes a variety of outcomes in mice, including respiratory, enteric, hepatic, and neurologic infections.

These infections often serve as highly useful models of disease. Interestingly, MHV-3 induces cellular injury through the activation of the coagulation cascade [ 68 ]. Most notably, A59 and attenuated versions of JHMV cause a chronic demyelinating disease that bears similarities to multiple sclerosis MS , making MHV infection one of the best models for this debilitating human disease.

Early studies suggested that demyelination was dependent on viral replication in oligodendrocytes in the brain and spinal cord [ 69 , 70 ]; however, more recent reports clearly demonstrate that the disease is immune-mediated.

Irradiated mice or immunodeficient lacking T and B cells mice do not develop demyelination, but addition of virus-specific T cells restores the development of demyelination [ 71 , 72 ]. Additionally, demyelination is accompanied by a large influx of macrophages and microglia that can phagocytose infected myelin [ 73 ], although it is unknown what the signals are that direct immune cells to destroy myelin.

These factors make MHV an ideal model for studying the basics of viral replication in tissue culture cells as well as for studying the pathogenesis and immune response to coronaviruses. Prior to the SARS-CoV outbreak, coronaviruses were only thought to cause mild, self-limiting respiratory infections in humans.

They cause more severe disease in neonates, the elderly, and in individuals with underlying illnesses, with a greater incidence of lower respiratory tract infection in these populations. HCoV-NL63 is also associated with acute laryngotracheitis croup [ 79 ]. One interesting aspect of these viruses is their differences in tolerance to genetic variability. HCoVE isolates from around the world have only minimal sequence divergence [ 80 ], while HCoV-OC43 isolates from the same location but isolated in different years show significant genetic variability [ 81 ].

Based on the ability of MHV to cause demyelinating disease, it has been suggested that human CoVs may be involved in the development of multiple sclerosis MS.

However, no evidence to date suggests that human CoVs play a significant role in MS. It is the most severe human disease caused by any coronavirus. The outbreak began in a hotel in Hong Kong and ultimately spread to more than two dozen countries. During the epidemic, closely related viruses were isolated from several exotic animals including Himalayan palm civets and raccoon dogs [ 82 ].

Although some human individuals within wet animal markets had serologic evidence of SARS-CoV infection prior to the outbreak, these individuals had no apparent symptoms [ 82 ]. Thus, it is likely that a closely related virus circulated in the wet animal markets for several years before a series of factors facilitated its spread into the larger population. Transmission of SARS-CoV was relatively inefficient, as it only spread through direct contact with infected individuals after the onset of illness.



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