Replication strategies of dna viruses

Once inside the nucleus and after association with host chromosomes, viral IN catalyzes insertion of viral sequences into the host DNA Fig. Integration of viral DNA e. A viral polyprotein is typically produced, which encodes the proteins required for replication. The replication process results in the formation of a dsRNA intermediate that is detected by the immune system.

Depending on the virus, sgRNAs may be generated during internal initiation on a minus-strand RNA template and require an internal promoter or there is the generation of a prematurely terminated minus-strand RNA that is used as template to make the sgRNAs. The resulting chimeric sg minus-strand RNA can in turn function as a template for the production of subgenomic positive-strand RNAs.

Translation of this mRNA generates proteins required for replication and viral encapsidation. As such, many dsRNA viruses undergo replication within their icosahedral capsids.

The replicating RNA polymerases are located within the capsid and produce mRNA strands that are extruded from the particle. Replication occurs in the cytoplasm. The viral RdRP complex is assumed to be the same for both replication and transcription and can switch off functions as required. Of note, two genome subgroups can be distinguished in this group: nonsegmented and segmented. Viruses with segmented genomes replicate in the nucleus, and the RdRp produces one monocistronic mRNA strand from each genome segment.

The mode of transcription is similar to eukaryotic transcriptional events in which the process is divided into three steps: 1 the initiation step, when a transcription initiation complex is assembled at the promoter region located upstream of the transcriptional start site, allowing for the recruitment of the RNA polymerase, 2 the elongation step, in which, the polymerase is recruited to template DNA, is activated by phosphorylation of the c arboxy- t erminal d omain CTD , and proceeds to transcribe the template DNA to RNA, and 3 the termination step, which involves the recognition of specific signals, including the polyadenylation signal.

For productive infection, viruses must then utilize this machinery, and remain both stable and undetected in the cell. Furthermore, while the great majority of cellular mRNAs are monocistronic, viruses must often express multiple proteins from their mRNAs. As a result, viruses have evolved a number of mechanisms to allow translation to be customized to their specific needs. Straightforward exploitation of the cellular capping machinery is typical of DNA viruses that replicate in the nucleus.

Other strategies used by viruses include internal initiation of translation of uncapped RNAs in picornaviruses and their relatives, snatching of capped oligonucleotides from host pre-mRNAs to initiate viral transcription in segmented negative-strand RNA viruses, and recruitment of genes for the conventional, eukaryotic-type capping enzymes that apparently occurred independently in diverse groups of viruses flaviviruses, reoviridae, poxviruses, asfarviruses, some iridoviruses, phycodnaviruses, mimiviruses, baculoviruses, nudiviruses.

For instance, flaviviruses e. Other examples that follow the same strategy include rotaviruses, barley yellow dwarf viruses, and possibly Hepacivirus C HCV. Since eukaryotic cells are not equipped to translate polycistronic mRNA into several individual proteins, DNA viruses overcome this limitation by using the cellular mechanism of splicing of their polycistronic mRNA to monocistronic mRNA.

RNA viruses, on the other hand, that mostly replicate in the cytoplasm, do not have access to these host mechanisms and consequently produce monocistronic sgRNAs e. However, the use of these mechanisms is not without consequences: 1 some viral proteins may be expressed from sgRNAs but the components of the replication complex that are needed early in infection must still be translated from the genomic RNA, 2 viruses with segmented genomes have to ensure the correct packaging of the different segments, and 3 polyprotein expression represents an inefficient use of host cell resources as all virus proteins are produced in equal amounts, even though catalytic proteins are often required in much smaller quantities than the structural proteins.

Alternative and more efficient mechanisms of expressing multiple proteins from a single viral mRNA involve internal ribosome entry, leaky scanning, ribosome shunting, reinitiation, ribosomal frameshifting, and stop codon read-through. Viral gene expression is facilitated by the possession of regulatory signals within viral mRNAs that are recognizable by the host cell. These signals ultimately enable the virus to shut off host gene expression to ensure preferential viral gene expression. The strategies are reviewed in the section that follows.

Transcription can be viewed as a highly regulated 3-phase process: initiation, elongation, and termination. Initiation of transcription requires the recruitment and assembly of a large multiprotein DNA-binding transcription initiation complex. During the course of evolution, several viruses have developed strategies that affect the loading of host transcription initiation factors into transcription complexes, which ultimately shuts down host protein synthesis Fig.

Viral mRNA transcripts compete against cellular mRNAs and preferentially gain access to the cellular gene expression machinery. Different strategies used by viruses to down regulate host transcription. The capping apparatus can be either host- or virus-derived. If virus-encoded, cleavage is carried out by the endonuclease activity of the viral RdRp.

Sequence complementarity shared between the nucleotides within the cleavage site of the donor mRNA and the viral RNA facilitates successful cap snatching. The cap-stealing mechanisms used by segmented RNA viruses to generate their mRNAs circumvent this innate detection system. Cap snatching of cellular mRNA. Downstream hairpin loops are RNA structures that facilitate initiation of cap-dependent translation in the absence of eIF2 translation initiation factors.

In addition, the physiological state of the infected cell dictates whether host mRNA transcripts undergo cap-dependent translation or cap-independent translation. When the cell exhibits normal housekeeping functions, translation of cellular mRNAs is carried out by a cap-dependent mechanism; however, under stressful conditions, such as heat shock, viral infection, hypoxia, and irradiation, the translation mechanism switches from cap dependency to IRES-driven mechanisms.

Infection by a range of viruses induces the activation of the ER stress response, resulting in the stimulation of IRES-dependent translation. As such, viruses containing IRES are able to efficiently benefit from the host cells ER stress response for their own multiplication. This is the site of binding of poly A binding protein in the cytoplasm. Viral mRNAs are synthesized without this signal sequence.

Stuttering occurs at a site containing a slippery sequence mononucleotide repeats and involves 1-base repeated frameshifts on the mRNA strand Fig. Stuttering mechanism. This mechanism, also observed in some eukaryotes, allows RNA viruses except dsRNA viruses to produce multiple proteins from a single gene. In these viruses, the RNA polymerase reads the same template base more than once, creating insertions or deletions in the mRNA sequence, thereby generating different mRNAs that encode different proteins.

There are two kinds of mRNA editing: 1 cotranscriptional editing through polymerase slippage and 2 posttranscriptional editing. RNA editing in members of the Ebolavirus genus increases their genome coding capacity by producing multiple transcripts encoding variants of structural and nonstructural glycoproteins from a single gene, ultimately increasing its ability for host adaptation. Also observed in many cellular organisms, alternative splicing allows production of transcripts having the potential to encode different proteins with different functions from the same gene Fig.

The sequence of the mRNA is not changed as with RNA editing; rather the coding capacity is changed as a result of alternative splice sites. Alternative splicing is regulated by cellular and viral proteins that modulate the activity of the splicing factors U1 and U2, both of which are components of the spliceosome.

Activation of the spliceosome is facilitated by cis-acting signals in the mRNA sequence. While only mature, spliced mRNA transcripts are exported out of the nucleus, hepadnaviruses and retroviruses are able to export nonspliced mRNA transcripts out of the nucleus for translation.

On the other hand, the NS1 protein n onstructural p rotein 1 of influenza viruses can interact with multiple host cellular factors via its effector- and RNA-binding domains. It is capable of associating with numerous cellular spliceosome subunits, such as U1 and U6 snRNAs, and can inhibit cellular gene expression by blocking the spliceosome component recruitment and its transition to the active state. Alternative splicing.

Alternative splicing is common in parvovirus pre-mRNA transcript processing and allows for the generation of different proteins from a specific nucleotide sequence on the viral mRNA strand. Dotted lines indicate alternative splice sites. Therefore, viruses can induce preferential induction of viral mRNA splicing by the cellular splicing machinery.

Knowledge concerning the coordination between cellular and viral genome splicing comes from adenoviruses and retroviruses, but only limited data are available for other viruses, for example, influenza viruses. This is also referred to as stop codon read-through, and is a programmed cellular and viral-mediated mechanism used to produce C-terminally extended polypeptides, and in viruses, it is often used to express replicases.

Termination of translation occurs when one of three stop codons enters the A-site of the small 40S ribosomal subunit. Stop codons are recognized by release factors eRF1 and eRF3 , which promote hydrolysis of the peptidyl-tRNA bond in the peptidyl transferase center P-site of the large ribosomal subunit. Read-through occurs when this leaky stop codon is misread as a sense codon with translation continuing to the next termination codon.

Read-through signals and mechanisms of prokaryotic, plant, and mammalian viruses are variable and are still poorly understood. Programmed ribosomal frameshifting is a tightly controlled, programmed strategy used by some viruses to produce different proteins encoded by two or more overlapping open reading frames Fig. Ordinarily, ribosomes function to maintain the reading frame of the mRNA sequence being translated. However, some viral mRNAs carry specific sequence information and structural elements in their mRNA molecules that cause ribosomes to slip, and then readjust the reading frame.

This ribosomal frameshift enables viruses to encode more proteins in spite of their small size. Ribosomal frameshifting. This occurs because the initiation codon can be part of a weak Kozak consensus sequence.

As a result, there can be the production of several different proteins if the AUG codon is not in frame, or proteins with different N-termini if the AUGs are in the same frame.

A number of viruses engage in leaky scanning, including members of the families Herpesviridae , Orthomyxoviridae , and Reoviridae. It is, therefore, referred to as cap-dependent discontinuous scanning. The mechanism of ribosome shunting has not been described in molecular detail. Shunting expands the coding capacity of mRNAs of viruses such as caulimoviruses. Ribosomal shunting. Ribosomes, therefore, skip the synthesis of the glycyl-prolyl peptide bond at the C-terminus of a 2A peptide cleavage of the peptide bond between a 2A peptide and its immediate downstream peptide.

Translation is then reinitiated on the same codon, which leads to production of two individual proteins from one open reading frame. Viruses not only employ strategies that maximize the coding capacity of their small genomes, disguise their mRNA with the same structural elements found in host mRNA, regulate their genome expression in a time- and space-dependent manner, but they have also evolved ways of subverting host cell functions in order to favor their own replication and translation.

These phosphorylation events serve to activate or deactivate the enzyme. Some viruses herpesviruses, bunyaviruses counteract this phosphorylation at serine amino acids to inactivate RNA polymerase, while other viruses orthomyxviruses, togaviruses disrupt cellular RNA polymerase function by signaling ubiquitination of the enzyme and its subsequent degradation by proteasomal action.

Phosphorylation of serine residues located on the CTD of the enzymes is blocked by some viruses. Other viruses arrest RNA Pol activity by signaling ubiquitination of the transcribing enzyme, which is subsequently degraded by the proteasome. Viruses can engage in targeted disruption of cellular mRNA export pathways to promote preferential viral gene expression Fig.

All DNA viruses replicate within the nucleus except poxviruses, asfarviruses, and phycodnaviruses. Updated from Baltimore D Bacteriol. Positive-strand viruses contain a single-stranded, message-sense RNA genome which is translated immediately after uncoating. To simplify this discussion, poliovirus family Picornaviridae ; genus Enterovirus will be used as a prototype because it is the most extensively studied positive-strand virus and provides a clear view of this strategy Fig.

Initial cleavage of the polyprotein occurs cotranslationally and is mediated by the autocatalytic activity of a virus-encoded protease, polypeptide 2A. Subsequent cleavages, catalyzed by viral protease 3C, yield structural capsid and catalytic polymerase proteins. Thus cap-independent initiation and internal ribosome entry allow poliovirus messages to be selectively translated under conditions where translation of capped messages is progressively compromised.

Following its synthesis, the viral RNA-dependent RNA polymerase catalyzes the synthesis of a full-length negative-sense copy of the genome. Subsequently, the negative-strand serves as template and directs the synthesis of multiple plus-strands.

Early in infection, when the concentration of viral structural proteins is low, newly synthesized positive-strands most likely are translated and serve to amplify the synthesis of viral proteins. Later when the concentration of virion precursors is high, newly synthesized plus-strands are encapsidated to generate infectious virus particles. VPg is thought to play a role in RNA synthesis, but it is unclear whether VPg functions as a primer or in some other capacity.

Because the genome of negative-sense RNA viruses cannot be translated, the first virus-specific biosynthetic event following uncoating is the synthesis of viral mRNA by a virion-associated RNA-dependent RNA polymerase using the viral genome as template.

Negative-stranded viruses are divided into two classes: those with unsegmented monopartite genomes i. Although each class uses the negative-strand strategy, they possess unique attributes and will be dealt with separately. The replication of monopartite viruses is discussed using vesicular stomatitis virus VSV , a rhabdovirus, as the prototype Fig. Transcription occurs within the nucleocapsid, a structure containing the viral genome and multiple copies of three virus-encoded proteins.

The polymerase, polypeptide L mol. The phosphoprotein P, present in about molecules per nucleocapsid, plays a variety of roles in RNA synthesis. It binds L to the nucleocapsid, maintains the solubility of free N and may function in chain elongation. After synthesis of the poly A tract, transcription terminates, releasing newly synthesized mRNA, but maintaining the transcriptase on its template. Re-initiation at the next gene downstream occurs via a conserved start sequence present at the beginning of each gene.

However, because re-initiation does not take place every time, downstream genes i. Thus viral gene expression is controlled by transcriptional polarity. Viral genome replication, i. The switch between the transcriptive and replicative modes appears to be controlled by the concentration of the nucleocapsid protein. When N reaches a critical concentration, it binds newly synthesized RNA within the leader sequence and allows the polymerase to readthrough intergenic regions and synthesize full-size positive-strands i.

Segmented viruses encode their genetic information in multiple molecules of negative-sense RNA. In the case of influenza A virus Orthomyxoviridae , the genome is composed of eight unique segments of virion RNA. In contrast to most RNA virus families, orthomyxoviruses require a functional cell nucleus for replication. This requirement reflects the fact that the orthomyxovirus polymerase complex can neither initiate transcription de novo nor cap and methylate viral mRNAs.

As with unsegmented viruses, the trigger controlling the transition from transcription to replication may be the concentration of nucleocapsid protein. Other negative-stranded viruses possess additional molecular surprises. Furthermore, in what may be the prototype of a new family within the Mononegavirales , Borna disease virus replicates and transcribes its genome within the nucleus and utilizes RNA splicing to generate its messages. Animal viruses with dsRNA genomes are segmented and can be viewed as a variant of the negative-sense strategy in which the virion encapsidates the replicative form of the genome.

Genomic dsRNA is transcribed within partially uncoated ribonuclease-resistant viral cores by the virion-associated polymerase to yield viral mRNAs.

Early in infection, some progeny plus-strands function as translational templates whereas others associate with nonstructural proteins and form complexes which are transcribed once to yield dsRNA.

Newly synthesized dsRNA serves as template for the synthesis of additional viral mRNA which amplifies the replication cycle. Later, as the concentration of core and capsid proteins increases, the dsRNA—protein complex exchanges nonstructural for structural proteins and forms mature virus particles.

After viral entry, the virion capsid is partially uncoated and a complementary DNA copy of the RNA genome is synthesized using reverse transcriptase.

An endonucleolytic activity, integral to the reverse transcriptase, degrades the RNA template and second-strand DNA synthesis begins. The upstream LTR plays a very important role in retrovirus gene expression because it contains enhancer elements which regulate pol II-catalyzed transcription. The downstream LTR is not involved in viral gene expression, but may activate host oncogenes and play a role in cell transformation.

Full-length genome-sized RNA can either be packaged within virions or serve as messenger for the capsid and catalytic viral proteins. Translation of retrovirus genomic RNA yields two classes of polyproteins. REV mediates the switch between the synthesis of regulatory proteins i. TAT and REV and the generation of structural and catalytic proteins by binding to cis -acting sequences within viral mRNA and directing the transport into the cytoplasm of unspliced genomic RNA and singly-spliced envelope message.

With the exception of parvoviruses and hepadnaviruses, the genomes of which are respectively single-stranded and partially double-stranded, DNA- containing animal viruses possess a dsDNA genome. In place of a detailed discussion of each family, broader issues of viral DNA replication will be discussed. To begin with, DNA viruses differ greatly in their genetic content ranging in size from 5 kbp Parvoviridae to greater than kbp Herpesviridae, Poxviridae and Iridoviridae.

Not unexpectedly, the degree to which virus replication is dependent on cellular functions reflects the genetic complexity of the virus. Thus, parvoviruses and papovaviruses require extensive host involvement to support viral biosynthetic events including DNA synthesis , whereas other families are progressively more independent.

Among herpes-, pox- and iridoviruses, viral genes are expressed in a coordinated temporal sequence of immediate early, early and late genes. Generally immediate early genes code for proteins required to initiate virus replication, early genes encode catalytic functions e.

Furthermore, immediate early genes activate early and late gene transcription, whereas specific early and late genes downregulate immediate early and early gene expression respectively. Aside from specific regulatory proteins, full late gene expression also requires viral DNA synthesis, thus inhibitors of viral DNA replication block late gene expression despite the presence of functional immediate early and early activators.

In contrast to other DNA viruses, hepadnaviruses possess a circular, partially single-stranded DNA genome that is replicated through an RNA intermediate using virus-encoded reverse transcriptase. The latter is encapsidated and transcribed into complementary DNA using virus-encoded protein P both as the primer and the reverse transcriptase. However, before its completion, the virion is exported from the cell leaving genomic DNA partially single-stranded.

Reflecting their metabolic independence from the host cell, poxviruses synthesize unique DNA and RNA polymerases, and their virions contain all the proteins needed to transcribe the earliest class of viral mRNAs. Furthermore, viral transcriptional promoters and termination sequences are unique and are regulated by virus-specific factors. Gene expression and DNA replication. The inverted terminal repeat ITR sequences at each extremity of the viral genome fold back on themselves, so that the free 3' OH group at the end of the virus single-stranded genome can be used as a primer for the synthesis of the complementary DNA strand.

The double-stranded parvovirus B genome contains a single promoter element that recruits cellular transcription factors and RNA polymerase. These viral proteins are all synthesized in the cytoplasm, then imported into the nucleus. NS1 is essential for replication of the virus genome — but it is not a DNA polymerase. Instead, NS1 acts as an origin recognition protein, which specifically binds to double-stranded virus DNA and allows host DNA polymerase to replicate viral DNA, generating many single-stranded copies of the parvovirus genome.

Parvovirus B19 can only replicate in actively cycling cells, that express all of the cellular factors necessary for DNA replication. Assembly and Release. Assembly occurs in the nucleus, and virions are released by cell lysis.

Otherwise, co-infection with either an Adenovirus or a Herpesvirus is necessary Adeno-Associated Viruses AAV developed for use as gene therapy vectors.

The Papillomavirus family was formerly grouped with the Polyomavirus family into the Papovavirus family PApilloma, POlyoma, simian VAcuolating virus 40 because members of both families have a similar structure.

However, it is now clear that the two families have a very different replication strategy and so each group has now been given its own family status.

They will not be discussed further in this section but see section of DNA tumor viruses. All have a similar strategy for DNA replication. Depending on the host cell, they can either transform the cell go here or replicate the virus and lyze the cell.

Attachment, penetration and uncoating. Viral capsid proteins interact with cell surface receptors and penetration is probably via endocytosis. Virions are transported to the nucleus and uncoated. DNA and associated histones enters nucleus, probably through a nuclear pore. Production of viral mRNAs and proteins. Gene expression is divided into early and late phases. Early genes encode enzymes and regulatory proteins needed to start viral replication processes.

Late genes encode structural proteins, proteins needed for assembly of the mature virus. Figure 3 Early gene expression Note: - - - - indicates regions of the primary transcript which are removed in the alternatively processed mRNA. Modified from Fiers et al.

Figure 4 Late gene expression Note: - - - - indicates regions of the primary transcript that are removed in the alternatively processed mRNA.

Broad arrows indicate regions translated into protein Modified from Fiers et al. Early gene expression figure 3 and 6. Post transcriptional RNA modification capping, methylation, polyadenylation, splicing etc. The early transcript primary transcript is made and then undergoes alternative processing, resulting in the mRNAs for the small T and large T antigens these proteins have common amino-termini but different carboxy-termini.

The mRNAs are translated in the cytoplasm. Note: Primary transcripts which can be processed and code for more than one protein are seen in several virus families and in the host cell.

By definition the late phase starts with the onset of viral genome replication. Large T antigen is needed for DNA replication. This binds to the origin of replication. Polyoma viruses use the host cell DNA polymerase, which recognizes the viral origin of replication if the T antigen is present. This process of DNA replication is very similar to that which occurs in the host cell - which is not surprising as the virus is using mainly host machinery except for the involvement of the T antigen.

Host histones complex with the newly made DNA. Late gene expression figure 4 and 6. Early mRNAs are still transcribed, but at a very much much lower rate. T antigen is involved in controlling increased transcription from the late promoter and decreased transcription from early promoter.

It also interacts with host proteins and changes the properties of the host cell, thus playing a role in cell transformation and tumor formation. VP1, 2, and 3 are made from same primary transcript which undergoes differential splicing figure 5.

Thus, one region of the DNA can code for two different amino acid sequences according to which reading frame is used. This is another way that viruses and cells can use a short stretch of DNA to code for more than one protein sequence.

VP1, 2 and 3 mRNAs are translated in the cytoplasm, the proteins are transported to nucleus, and capsids assemble with DNA and cell histones inside the capsid. Large numbers of capsids accumulate in the nucleus and form inclusion bodies.

Virions are released by cell lysis. Figure 5 VP1, 2, and 3 are made from same primary transcript which undergoes differential splicing. Early genes are in red, late genes are in green. Note: - - - - indicates regions of the primary transcript which are removed in the alternatively processed mRNA.

Cross-hatched area indicates region of RNA translated in different reading frames according to which alternatively spliced transcript is being translated Modified from Fiers et al. Adsorption and penetration. Adenoviruses usually infect epithelial cells. The fibers bind to a cell surface receptor and the virus is engulfed by endocytosis. The virus appears to be able to lyze endosomes. Uncoating occurs in steps. DNA is released into the nucleus probably at a nuclear pore figure 9. Early transcription: Adenovirus uses host cell RNA polymerase and early mRNAs are transcribed from scattered regions of both strands figure Multiple promoters result in more flexible control.

Adapted from Zinsser Microbiology 20 th Ed. Figure 10 Transcription map of adenovirus. Early genes are shown in red.

Black indicates late genes. Blue lines indicate DNA. Square brackets indicate the positions of promoters. Primary transcripts are made from each promoter and then undergo alternative splicing, the diagram above does not show the primary transcript.

It only shows those regions present in the alternatively spliced products. Missing regions indicate removal of introns. Adapted from Broker, T. In Processing of RNA. Adenovirus encodes its own DNA polymerase which is one of the early proteins. The DNA is replicated by a strand displacement mechanism figure There are no Okazaki fragments , both strands are synthesized in a continuous fashion.

DNA polymerases cannot initiate synthesis de novo, they need a primer. In the case of adenovirus, the virally coded terminal protein TP acts as a primer. It is thus found covalently linked to the 5' end of all adenovirus DNA strands. Figure 12 Processing of viral primary transcript Late transcription.

The way in which late transcription is switched on is not well understood. The primary transcript is processed to generate various monocistronic mRNAs figure 12 :. There are two types of cleavage of primary transcript:. It is not understood how this process is controlled such that the correct amounts of each mRNA are made.