Replication in rna virus

Furthermore, clinical trials have been conducted for several indications with self-amplifying RNA viruses. In this context, alphaviruses have been subjected to phase I clinical trials for a cytomegalovirus vaccine generating neutralizing antibodies in healthy volunteers, and for antigen delivery to dendritic cells providing clinically relevant antibody responses in cancer patients, respectively.

Rhabdoviruses have generated promising results in phase III trials against Ebola virus. 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. Few RNA viruses, including bornaviruses, orthomyxoviruses, and retroviruses, replicate in the nucleus. Trafficking between the nucleus and cytoplasm is usually unidirectional for large macromolecules like the mRNA transcript, and occurs through the n uclear p ore c omplex NPC.

Viruses that replicate in the nucleus must out-compete cellular mRNAs to export viral mRNAs out of the nucleus for translation into virus gene products in the cytoplasm.

Several viruses can inhibit nuclear export of cellular mRNAs by disrupting nuclear export receptors exportin1 and TIP-associated protein and nucleoporins that comprise the NPC to compromise their function in nucleocytoplasmic trafficking of cellular mRNA. One half of the NPC is shown in the diagram. Many DNA viruses e. Viruses have developed different strategies to effectively degrade host mRNAs and to allow preferential translation of their own mRNA Fig.

Most viruses produce an endonuclease that cleaves host mRNAs, which are then degraded by host exonucleases e. Betacoronaviruses, influenza viruses, vaccinia viruses, and herpesviruses can produce viral endonucleotyic products to an extent that saturates cellular RNA decay-related quality control mechanisms and limit their function.

Transcripts of cytoplasmic viruses must circumvent the cellular mRNA decay machinery to enable virion production. Picornaviruses are able to suppress cellular RNA decay factors, and polioviruses and human rhinoviruses produce viral proteases that degrade Xrn1, Dcp1, Dcp2, Pan3 a deadenylase , and AUF1decay factors. Viruses capable of inducing the shutdown of cellular mRNA translation are able to continue to translate at least part of their mRNAs using noncanonical translation mechanisms, for example, cap-independent translation, ribosome shunting, and leaking scanning e.

Shutoff of host translation machinery by viral interference with specific eukaryotic translation initiation factors and poly A binding protein PABP. Most viruses interact with cellular chaperones in order to ensure correct folding of viral proteins.

Viral proteins often consist of multiple domains or are produced as polyprotein precursors, which must be processed before they can be functional. The coat protein or capsid is a meta-stable structure that must be specifically assembled in a preordered arrangement without reaching minimum free energy; yet must be disassembled upon entry of the host cell.

Some cellular chaperones, for example, Hsp70, are used to accelerate the maturation of viral proteins and are involved in regulating the viral biological cycle. The high rate of mutation in RNA viruses may mean an increased dependency on chaperones for the gene products of these viruses. Hsp70 can refold denatured proteins, which negates some of the destabilizing alterations in structural proteins as a result of mutated genes.

This ensures that a high proportion of viral proteins is accurately configured to function in virus multiplication. Viruses can manipulate the cellular metabolism to provide an increased pool of molecules, for example, nucleotides and amino acids, which are required for viral gene expression and virion assembly. Some viruses need to create a lipid-rich intracellular environment favorable for their replication, morphogenesis, and egress. Replication of HCV occurs on specific lipid raft domains, whereas assembly occurs in lipid droplets.

As such, in order for HCV to create replication compartments and increase sites of assembly, the RNA virus requires both the synthesis of fatty acids, for example, cholesterol, sphingolipids, phosphatidylcholine, and phosphatidylethanolamine, and formation of lipid droplets.

Lipids are especially required for assembly of virions of enveloped viruses as these molecules are a major component of membranes. Cellular lipid metabolism is affected at three levels: enhanced lipogenesis, impaired degradation, and disruption of export, which is subsequently manifested in the host as HCV-associated pathogenesis.

Viral interference of the host cell cycle can result in the dysregulation of cell cycle checkpoint control mechanisms to promote viral replication and to facilitate efficient virion assembly.

Both DNA and RNA viruses specifically encode proteins responsible for targeting and arresting essential cell cycle regulators to create intracellular conditions that are favorable for viral replication and propagation.

Retroviruses and other RNA viruses also interfere with the host cell cycle. Viral-mediation of the cell cycle can increase the efficiency of viral gene expression and virion assembly. Cell cycle arrest may delay apoptosis of infected cells.

Many viruses encode a cyclin-D homolog protein v-cyclin that associates with Cdk6 to phosphorylate Rb, which regulates G1 phase. Various DNA viruses primarily infect quiescent or differentiated cells, which contain low levels of deoxynucleotides dNTPs as these cells do not undergo active cell division. As such, a restricted pool of dNTPs will not provide an ideal environment for viral replication. It has been proposed that such viruses can induce quiescent cells to enter the cell cycle, specifically the S phase, in order to create an environment that generates factors, such as nucleotides, that are required for viral replication.

This specificity determines the host range tropism of a virus. Penetration: The process of attachment to a specific receptor can induce conformational changes in viral capsid proteins, or the lipid envelope, that results in the fusion of viral and cellular membranes. Some DNA viruses can also enter the host cell through receptor-mediated endocytosis. Uncoating: The viral capsid is removed and degraded by viral enzymes or host enzymes releasing the viral genomic nucleic acid. Replication: After the viral genome has been uncoated, transcription or translation of the viral genome is initiated.

It is this stage of viral replication that differs greatly between DNA and RNA viruses and viruses with opposite nucleic acid polarity. This process culminates in the de novo synthesis of viral proteins and genome. Assembly: After de novo synthesis of viral genome and proteins, which can be post-transrciptionally modified, viral proteins are packaged with newly replicated viral genome into new virions that are ready for release from the host cell.

This process can also be referred to as maturation. Rep-TD1c derivatives containing different potential RE riboswitches in place of the SL5 RE were analyzed in vivo for replicon responsiveness to theophylline. Cells were cotransfected with in vitro -generated RNA transcripts of helper sg1T viral genome and different riboswitch-containing replicons and then incubated in liquid medium in the presence or absence of theophylline.

Three representative examples from a larger pool of candidates that were screened data not shown are presented in Fig. Results from the replicon analysis were divided into three categories based on theophylline responsiveness and replicon accumulation: i non- or weakly responsive with constitutively high levels of replicon accumulation, ii non- or weakly responsive with constitutively low levels of replicon accumulation, or iii responsive with inducible replicon accumulation.

Rep-2A iii showed a notable increase in accumulation in the presence of 0. The notable induction observed for Rep-2A indicated that an appropriate balance of stability for the unbound aptamer was achieved; it was unstable enough to inhibit viral RNA replication, yet ordered enough to bind to its ligand.

Next, a ligand dose—response assay was carried out to assess the effectiveness of induction. Maximal induction was achieved with 0. To determine whether the observed induction required a functional aptamer, a replicon containing a mutated aptamer was tested Fig. Rep-2Am, in which an essential nucleotide in the theophylline-binding pocket was substituted 16 , was not responsive to theophylline Fig.

In addition, the necessity and specificity of theophylline for induction was evaluated by using caffeine as a ligand. Caffeine is a molecular analogue of theophylline that differs in that it contains a methyl group at N7 Fig. This structurally similar ligand was not able to activate replication of Rep-2A Fig. Collectively, these analyses indicate that the induction of viral RNA replication is aptamer- and ligand-dependent, as well as theophylline-specific and ligand-dose-dependent.

Analysis of an RE riboswitch in a viral replicon. A The effect of increasing concentrations of ligand theophylline or caffeine on the relative levels of Rep-2A or Rep-2Am accumulation in plant-cell protoplasts at 22 h after cotransfection.

C Induction of Rep-2A accumulation at various times after cotransfection. Effective induction was also observed when theophylline treatment was delayed for up to 6 h after cotransfection of viral RNAs note that Rep molecules are very stable within cells 15 Fig. This result demonstrates that ligand treatment does not have to immediately follow transfection of its target RNA and suggests that riboswitch-containing viral RNAs that are expressed constitutively in cells at low levels from either a DNA vector or a genome-incorporated transgene would also be inducible by using this approach.

Indeed, the ability to control both the timing and level of activation provides a powerful combination for modulating a viral process. Next, theophylline-based induction of replication was tested in the more complex and biologically relevant context of the TBSV genome.

Additional screening of a small group of viral genomes that contained aptamers with different closing stems in place of SL5 allowed for identification of one, GenA, that was suitably responsive Fig. RE-riboswitch-containing viral genomes were transfected into plant-cell protoplasts, incubated in the absence or presence of theophylline 0. GenA shaded was identified as the most responsive to theophylline.

B Dose-dependent theophylline induction of GenA. Protoplasts were transfected with GenA and incubated in the presence of different concentrations of theophylline. GenA accumulation levels were quantified by Northern blot analysis at 22 h after transfection. To gain possible insight into the function of the SL5 RE in genome replication, the accumulation levels of both plus and minus strands of viral RNAs, including subgenomic sg mRNAs, were analyzed.

The corresponding levels of minus-strand sg mRNAs for GenA were also similar to those of the control genome, but the minus-strand genome level was greater than that of the control genome Fig. With increasing concentrations of theophylline, the relative plus-strand genome levels were markedly restored, with sg mRNA levels increasing moderately Fig.

In contrast, the corresponding minus-strand genome and sg-mRNA levels were much less affected by changes in theophylline concentration Fig. The concentration of theophylline that transfected plant-cell protoplasts were incubated in posttransfection is indicated at the top, and the positions of the genome g and sg mRNAs sg1 and sg2 are indicated to the left.

The TBSV genome is shown in the middle. Synthesis of progeny genomes, i. These findings are significant in several respects. First, they indicate that SL5, which functions as a plus-strand RE in the genome 15 , is required for efficient accumulation of progeny viral genomes.