Virus synthesize

The synthesis of artificial viruses has been discussed for such applications as pollution reduction and advanced filtering. Specialized microbes can be created to consume almost anything. On the other hand, there is always the risk that a malicious party would use this technology to create a virus engineered for high virulence or lethality against human hosts.

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Login: Forgot password? They associate with the genome through interactions with RNA-binding nucleocapsid N or capsid proteins. Therefore, naked purified away from protein genomic RNA is not infectious, cannot be translated, and will eventually be degraded if transcription is blocked. Before genome replication can proceed, viral mRNAs must be transcribed and translated. If purified virions are gently lysed under appropriate buffer conditions, with the addition of NTPs, mRNAs will be transcribed in the test tube.

However, genome RNA will not be synthesized under these conditions Table Schematic representation of replication of genomes of minus-strand RNA viruses. Upon entry into the cell, the active transcription complex synthesizes mRNAs. This process can also occur in a test tube see Fig. Translation of mRNAs produces proteins required for genome replication. Thus if protein synthesis is blocked in the infected cell, mRNAs continue to by synthesized but genome replication does not occur.

Newly synthesized proteins provide the switch from transcription to genome replication. The genomes of viruses in the order Mononegavirales are unsegmented, negative-strand RNA. Note that genome synthesis does not occur under these conditions. In contrast, viruses that use an ambisense coding strategy transcribe some mRNAs from the copy genome.

There are virus families in which some members are considered negative-strand RNA viruses while others use an ambisense strategy. Thus these two strategies are closely related. Some ambisense viruses package copy genomes that can be used as templates for transcription, such that the full complement of viral genes can be transcribed soon after infection. It should be noted that packaged copy genomes are not mRNAs and are not translated. Reoviruses also have segmented genomes, packaging 11—12 segments of dsRNA.

Reoviruses are nonenveloped and particles consist of two or three concentric icosahedral capsid layers. A unique feature of the reovirus replication cycle is that the genome segments are transcribed from within the capsid. The genomes of RNA viruses have some common general features. Obviously there are one or more open reading frames that encode the viral proteins. But there are also regions of RNA that do not code for protein. These non-coding regions NCRs or untranslated regions UTRs are often highly conserved within a virus family, indicating that they have important functions.

NCRs may have specific, critical nucleotide sequences but in some cases they are regions of the genome that fold into conserved structures, and structure may be more critical than a specific sequence. Of course a source of RdRp must be supplied. RdRp may be encoded in the minigenome or may be supplied in trans by using a cell line stably expressing the viral RdRp, for example. The sequences required to direct RNA replication are often fairly simple and can be linked to virtually any RNA sequence to drive its replication.

These promoter sequences can be rather short but provide a means to direct the RdRp to internal sites on the genome. There may also be specific RNA sequences that signal polyadenylation. There are a variety of different strategies that RNA viruses use to regulate transcription and genome replication, but all involve RNA sequences found in the genome.

The RNA genomes of some viruses are highly structured and extensively base paired. The IRES serves as a platform for ribosome assembly. Promoters can be quite long and complex and promoter regions themselves are not transcribed.

It is particularly important, in the case of genome synthesis, that genetic information not be lost or modified; however, mRNAs are often capped and polyadenylated. Are the methods for priming viral mRNA synthesis the same or different from the methods of priming genome replication?

The RNA viruses seem to have experimented widely. For example, the picornaviruses use poly A tracts encoded in the genome. Among the negative-strand RNA viruses, those in the order Mononegavirales use a stuttering mechanism to synthesize long poly A tracts from short poly U tracts Fig. A strategy to regulate mRNA synthesis. This figure shows the organization of a paramyxovirus genome paramyxoviruses are members of the order Mononegavirales ; negative-strand RNA viruses with unsegmented genomes.

Each protein-coding region is flanked by regulatory sequences that control capping and polyadenylation. The order of the genes on the genome regulates the relative quantities of mRNAs synthesized. Because RdRp does often dissociate from the genome during transcription, the downstream genes are produced in lower quantities. Even with fairly simple genomes, RNA viruses must, and do, regulate the amounts of genome, copy genome, and mRNAs that are synthesized during an infection. It is much more efficient to synthesize many genomes from each copy genome.

Internal promoters for mRNA synthesis can vary in sequence, controlling the relative affinity of the transcription complex for each mRNA. Thus, whereas picornaviruses impair cap-dependent translation by reducing the abundance of full-length initiation factors, HCMV stimulates the cap-dependent translation machinery and increases initiation factor concentration.

How this is achieved and its contribution to viral replication remain unknown. Nevertheless, raising the concentration of eIF4F subunits may potentiate complex assembly. Instead of increasing host initiation factor levels, mimiviruses — large 2 Mb DNA viruses that infect Acanthamoeba spp. However, the capacity of mimiviral homologues to function in translation initiation and their contribution to protein synthesis in infected cells are unknown. Although their abundance remains constant, changes in the local concentration of translation factors probably regulate protein synthesis in poxvirus- or asfarvirus-infected cells.

Increasing the local concentration of initiation factors in discrete regions could favour eIF4F assembly and sequester factors from host mRNAs to suppress cellular translation. For instance, eIF4E is redistributed to the nucleus by poliovirus 61 , possibly helping to suppress host translation. Interestingly, these viruses impair host protein synthesis. VacV replication is similarly reduced in MNK1-deficient cells Although MNK-dependent eIF4E phosphorylation is not absolutely required for protein synthesis and is poorly understood, it is associated with increased viral protein synthesis and viral replication in herpesvirus-, asfarvirus- and poxvirus-infected cells.

Excluding the mRNAs of cricket paralysis virus CrPV , which dispense with initiation factors 67 , 68 , most viral mRNAs recruit 40S subunits directly or indirectly through eIF3, irrespective of their requirement for eIF4F or their use of cap-dependent versus cap-independent mechanisms. In fact, viruses that rely on cap-independent translation to circumvent eIF4F often target eIF3 and ribosomal proteins.

Finally, eIF3 is targeted both by viruses seeking to inhibit host protein synthesis and by host defences attempting to impair viral protein production. Interfering with eIF3 via viral proteins and host antiviral functions can suppress protein synthesis in cells infected with RNA viruses.

Host antiviral defences also target eIF3. How cellular eIF3-inhibitory functions are controlled once they are produced is not known. Importantly, the process of ternary-complex formation and 40S loading is targeted by an innate host response designed to globally inhibit protein synthesis in virus-infected cells.

Although activation of any eIF2 kinase in virus-infected cells could inhibit protein synthesis and potentially result in autophagy 82 , type I interferon production by virus-infected cells stimulates PKR accumulation in neighbouring cells.

PKR activation following infection of interferon-primed neighbouring cells globally inhibits protein synthesis and curtails viral spread, making this activation a key player in the innate response to viruses 1 , However, host efforts to inactivate eIF2 by phosphorylation in order to limit viral replication are matched by viral countermeasures to inhibit interferon production and therefore indirectly prevent PKR accumulation, to directly preserve eIF2 activity for viral protein production or to bypass eIF2 function entirely Fig.

The resulting 80S ribosome carries out the elongation phase Fig. Preserving eIF2. Inactivating eIF2. Some viruses benefit from eIF2 inactivation. Whereas translation initiation is rate limiting and involves numerous factors that are each subjected to intricate regulation, the processes of elongation and termination require a more limited set of factors, but viruses can nonetheless effectively target these factors.

Increased elongation rates are required to cope with elevated initiation rates. Viruses can alter eEF function and subcellular distribution. Similarly, viral manipulation of termination factors can regulate polyprotein synthesis or couple termination to re-initiation. Although most translational control strategies operate at the rate-limiting initiation step, different regulatory mechanisms target elongation and termination.

Translation elongation begins after eukaryotic translation initiation factor 5B eIF5B -mediated 60S subunit joining triggers eIF release, and the assembled 80S ribosome begins polypeptide chain extension. Ribosome-catalysed peptide bond formation precedes eEF2-mediated translocation of the peptidyl-tRNA into the P site and the de-acylated tRNA into the E site, exposing the unoccupied A site for successive rounds of elongation that form the polypeptide chain.

Phosphorylation by eEF2 kinase inhibits eEF2 activity. Viral functions that regulate elongation are indicated; see main text for details and abbreviations. Pi, inorganic phosphate. Murine norovirus VP2 is synthesized by such coupling Retroviral reverse transcriptase binds eRF1 to modulate termination and re-initiation , and re-initiation protects HIV-1 mRNAs from nonsense-mediated decay Finally, termination in small uORFs can have a regulatory role by restricting scanning ribosomes from re-initiating at downstream cistrons.

Ultimately, the stalled ribosome disengages the mRNA On recognition of a stop codon in the A site, eukaryotic release factor 1 eRF1 triggers 80S arrest and polypeptide release. Virally encoded functions that regulate termination are indicated. Notably, HIV reverse transcriptase and the termination upstream ribosomal-binding site TURBS RNA cis -elements in influenza B virus and feline calicivirus FCV allow eukaryotic ribosomes to efficiently re-initiate translation, a property normally associated with prokaryotic ribosomes.

Competition between virus and host for limiting translation components is influenced by mRNA availability in the cytoplasm of the infected cell. Viruses can interfere with mRNA trafficking, altering mRNA steady-state levels to impair host protein synthesis while stimulating the cellular translation machinery. Other viruses that replicate in the nucleus, such as adenoviruses or influenza viruses, also inhibit host mRNA export , How influenza virus mRNAs reach cytoplasmic ribosomes remains unknown, although the TAP nuclear export pathway is required Instead of a ribonuclease, poxviral decapping enzymes remove m 7 G caps from mRNAs, converting them into substrates for host mRNA decay pathways and contributing to host shut-off 4.

Viral infection alters the distribution and composition of stress granules and processing bodies P-bodies , which are discrete cytoplasmic structures associated with mRNA metabolism Poliovirus in particular degrades factors involved in P-body formation poly A -nuclease and the exonuclease XRN1 and induces formation of modified stress granules that lack G3BP, which is cleaved by the viral 3C protease , , Although the function of modified stress granules in infected cells remains unclear, inactivating P-body components might protect viral mRNAs, which are uncapped, from degradation.

By contrast, stress granule induction by reoviruses may inhibit host translation Although stress granule components can stimulate replication of some RNA viruses respiratory syncytial virus, dengue virus and West Nile virus , other viruses exploit P-bodies for viral assembly HIV-1 and brome mosaic virus 96 , or cap stealing hantaviruses 7.

Although RNAi is a potent host antiviral defence mechanism in plants and invertebrates, virally encoded functions can antagonize the host miRNA machinery. Whether miRNAs contribute to mammalian innate antiviral responses remains less clear.

However, herpesviruses in particular do express virally encoded miRNAs in latently infected cells, and these miRNAs are thought to suppress expression of lytic genes and help maintain latency.

They also suppress host apoptotic and immune responses. This may reflect their cytoplasmic replication, which could restrict access to nuclear miRNA-processing steps, or, for some RNA viruses, may reflect the potentially detrimental effects of miRNA processing on viral genome integrity.

Induction of a host transcription factor in enterovirus-infected cells promotes miR expression, which impairs translation of eIF4E-encoding mRNA and inhibits cap-dependent protein synthesis Indeed, much remains to be learned about miRNA targets and their contribution to infection. The effectiveness with which viruses co-opt components of the host translation machinery represents an extraordinary example of parasitism and illustrates the importance of this process to viral replication.

Nature validates this view, as initiation factors determine plant susceptibility to RNA viruses. Virus—host interactions that regulate protein synthesis in infected cells could potentially lead to novel broad-spectrum antiviral targets that are ripe for development.

Even antagonizing a general factor such as eIF4F may be tolerated for limited periods to combat acute, life-threatening infections, as high eIF4F activity seems to be reserved for translating complex, growth-related mRNAs.

The MNK proteins are interesting targets, as they are not essential, core initiation factors but instead have a regulatory role.

Other targets, such as inhibitors of the mTOR active site which disrupt eIF4F and impair herpesviral replication 43 , will probably have immunosuppressive side effects in vivo Finally, virus—host interactions that regulate translation have contributed to the development of oncolytic viruses Viruses subvert nearly every step in the host translation process.

From mRNA availability for cytoplasmic ribosomes, to cell-signalling pathways that regulate translation factor abundance, localization and activity, to ribosome recruitment, all are commandeered to stimulate and sustain viral mRNA translation. The diversity of strategies used by different viruses reflects the varied viral life cycles, the specialized host cells that viruses infect and the methods of translation control in their cellular hosts which are probably the main evolutionary drivers behind the diverse strategies used for subversion.

Similarities between the translation control strategies that are operative in infected cells and in stress-induced, uninfected cells have emerged. Adenovirus-infected and uninfected, heat-stressed cells use ribosome shunting. Key integrators such as TSC and mTORC proteins, which enable rapid control of cap-dependent translation in response to physiological cues in uninfected cells, have important roles stimulating or repressing translation in virus-infected cells.

IRESs were originally discovered as viral genetic elements, but they enable translation of cellular mRNAs when eIF4F-mediated, cap-dependent translation is impaired by stress. By conferring eIF2 independence, newly identified factors such as ligatin could expand the range of conditions that support viral mRNA translation. Finally, IRESs with minimal initiation factor requirements such as those of HCV and CrPV highlight how viral models provide powerful cell-biological and genetic tools that continue to expose surprising translation regulatory mechanisms.

There are four types of internal ribosome entry sites IRESs see the figure , each of which can directly interact with host translational components and circumvent conventional cap-dependent ribosome recruitment These sites can confer a potent competitive advantage to viral mRNAs, freeing them from host regulatory constraints and, in cases for which viral infection impairs cap-dependent translation, sustaining viral protein synthesis.

Remarkably, these IRESs interact with the 40S subunit directly, inducing conformational changes and facilitating 60S subunit joining to form 80S ribosomes independently of initiation factors. An initial 'pseudo-translocation' of the Ala-tRNA to the P-site initiates translation, resulting in viral precursor polypeptides with amino-terminal alanine residues rather than methionine.

Differences in the physical structure, ORF organization, ribosome composition and initiation factors for bacterial mRNAs compared with eukaryotic mRNAs influence bacteriophage translation strategies Ribosomes also re-initiate translation efficiently in bacteria, enabling the translation of polycistronic mRNAs.

This allows RNA replicases and coat proteins to suppress translation, fostering genome replication and RNA packaging, respectively. Phage T4 proteins involved in DNA replication autogenously repress translation of their own encoding mRNA by sequence-specific or, in the case of gp32 which binds single-stranded DNA , cooperative structure-specific binding.

Restricting the repressor activity of gp32 to unbound monomers that are superfluous for DNA replication serves as a rheostat, limiting gp32 accumulation Similarly, translation repression by free, unassembled phage P22 gene 8 scaffold protein maintains the scaffold-to-coat protein ratio that is required for phage assembly Processing of phage T7 1. Transit through an upstream cistron by translating ribosomes can modify the higher-order structure of a polycistronic transcript, regulating initiation for the downstream cistron by controlling SD exposure Such 'translational coupling' requires ribosome release factors when translation of a downstream cistron involves an upstream ribosome that must terminate before re-initiating compared with entry of a new ribosome Besides coupling, other methods of maintaining subunit stoichiometry are recoding and bypassing.

These processes also maximize coding capacity by altering ribosome decoding of a contiguous ORF. Bypassing joins the information in two ORFs into one polypeptide. Translation control provides a powerful physiological sensor that regulates the lytic phase—lysogenic phase developmental decision in temperate phages.

Even more elaborate systems are found in phage P1 and phage P7, in which prophage C4 antisense RNAs indirectly antagonize the synthesis of anti-repressor by combining translational repression and coupling to regulate transcription Bacterial antiviral responses also exploit the translation control mechanisms of phages. A similar motif in the AbiD1 gene of the host, Lactococcus lactis , confers Orf1 responsivenes s and results in an abortive infection Jackson, R.

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