MicrobiologyBytes

A range of mechanisms exist to enable maintenance and restoration of genome integrity in RNA viruses. Despite their often fastidious mechanisms to ensure accurate replication initiation and termination, virus RNA-dependent RNA polymerases (RdRps) are sufficiently flexible to accommodate alternative modes of initiation and elongation, enabling terminal repair, terminal transferase activity and recombination. For any given virus, the behaviour of the RdRp at the 3′ end of a template might be impacted by the nature of the 3′-terminal sequence. It seems likely that, if the genome termini are intact and all promoter and accessory sequences required for replication initiation are present, the RdRp will engage preferentially in accurate replication initiation. However, if key terminal sequences are missing, the replicase complex might not be able to assemble correctly, releasing the RdRp to perform ‘abnormal’ actions, such as non-templated polymerization or terminal transferase activity. Once a genome has been repaired, the RdRp could revert to its ‘normal’ function of template-dependent polymerization.

Viruses that replicate in particularly harsh environments or which have no passive defences to protect their genomes may have replicases that accommodate alternative initiation mechanisms to facilitate terminal repair more readily, and fundamental differences in virus genome and replicase architecture probably affect the propensity for RNA recombination. It is striking that most examples of RNA virus genome repair involve positive-strand viruses, whose genomes might be more vulnerable than those of the double-stranded or negative-sense viruses, in which the genomes are sequestered in protein capsids during the entire cycle of infection. The data also suggest that formation of truncated genomes, whilst hindering virus replication kinetics, might allow or aid some viruses to become persistent, which ultimately could aid their propagation within the host population. Thus the capacity for genome repair could be an important factor in virus pathogenesis.

How RNA viruses maintain their genome integrity. 2010 J Gen Virol. 91(6): 1373-1387
RNA genomes are vulnerable to corruption by a range of activities, including inaccurate replication by the error-prone replicase, damage from environmental factors, and attack by nucleases and other RNA-modifying enzymes that comprise the cellular intrinsic or innate immune response. Damage to coding regions and loss of critical cis-acting signals inevitably impair genome fitness; as a consequence, RNA viruses have evolved a variety of mechanisms to protect their genome integrity. These include mechanisms to promote replicase fidelity, recombination activities that allow exchange of sequences between different RNA templates, and mechanisms to repair the genome termini. In this article, we review examples of these processes from a range of RNA viruses to showcase the diverse approaches that viruses have evolved to maintain their genome sequence integrity, focusing first on mechanisms that viruses use to protect their entire genome, and then concentrating on mechanisms that allow protection of the genome termini, which are especially vulnerable. In addition, we discuss examples in which it might be beneficial for a virus to ‘lose’ its genomic termini and reduce its replication efficiency.

Related:

• Negative sense RNA viruses
• Expression strategies of ambisense viruses
• RNA replicons – a new approach to influenza virus vaccines
• Three-dimensional analysis of a virus RNA replication reveals a virus-induced mini-organelle

The RNA silencing based antiviral plant response is one of the best studied antiviral strategies in plants. The key element of RNA silencing based antiviral strategies is the virus derived small interfering RNA (vsiRNA), which guides the RNA induced silencing complex (RISC) to target viral genomes in plants and invertebrates. siRNAs are processed from double-stranded RNAs (dsRNA) or structured single-stranded RNAs (ssRNAs) by RNase III-like enzymes such as DICER (in plants there are several Dicer-like genes). siRNAs guide the sequence-specific inactivation of target mRNAs by RISC. Plant RNA viruses are strong inducers as well as targets of RNA silencing and high levels of vsiRNAs accumulate during the viral infection. However, despite of the extensive studies of siRNA biogenesis the origin of plant viral siRNA is still not understood. vsiRNAs are thought to be processed from ds viral RNA replication intermediates, local self-complementary ds regions of the viral genome or through the action of RNA-dependent RNA polymerases on viral RNA templates. In plants two distinct classes of vsiRNAs have been identified: the primary siRNAs, which result from DCL mediated cleavage of an initial trigger RNA, and secondary siRNAs, whose biogenesis requires an RDR enzyme.

Researchers profiled Cymbidium ringspot virus (CymRSV) derived short RNAs using three different methods. Profiling of viral short interfering RNAs revealed a different sequence bias for the 454 and Solexa high-throughput sequencing platforms. They found that viral short RNAs are primarily produced from the positive strand of the virus and produced with very different frequency along the viral genome. The hybridisation approach showed that the profile of viral short RNAs is determined by the virus itself because the profiles were the same in different species and it also showed that the process was RDR6 independent. These results suggest that CymRSV short RNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase. Regions from the viral genome that are not complementary to highly abundant viral short RNAs were targeted in the plant just as efficiently as regions recognised by abundant short RNAs.

Structural and Functional Analysis of Viral siRNAs. 2010 PLoS Pathog 6(4): e1000838. doi:10.1371/journal.ppat.1000838
A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.

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• Resistance to Plant Viruses
• A cream that could stop genital herpes?

Influenza A virus is the prototype of the Orthomyxoviridae, and like all members of this family, the negative-sense RNA that comprises its genome is divided into separate segments. These vRNA segments share a common organisation; a long central coding region (in antisense), sometimes encoding more than one polypeptide, flanked by relatively short untranslated regions (UTRs) and at the termini, sequences conserved between segments that show partial complementarity. The vRNA segments are separately encapsidated into ribonucleoprotein (RNP) structures by viral polypeptides. These RNPs act as independent units for the purposes of viral RNA synthesis, which occurs in the nuclei of infected cells. Replicated vRNAs are exported (as RNPs) from the nucleus via the cellular CRM1 pathway, and at the final stage of viral assembly, are incorporated into the virion as it buds from the apical plasma membrane of the cell. The process of virion assembly is not well understood but is thought to involve a series of protein-protein interactions between the cytoplasmic tails of the viral integral membrane proteins, the matrix protein and the RNPs.

Genome segmentation confers evolutionary advantages on influenza viruses, but also poses a problem in virion assembly. The eight segments encode 12 identified polypeptides. At least one copy of each of the eight vRNAs must be packaged for a single virion to be able to initiate a productive infection. Until recently, the process by which this was achieved was poorly understood, but a clearer picture has begun to emerge of a mechanism for specifically packaging a full genome, mediated by cis-acting packaging signals in the vRNAs. This review aims to summarise the thought processes and experimental evidence leading up to the currently accepted model for influenza A genome packaging and to highlight the main questions remaining.

Genome packaging in influenza A virus. J Gen Virol. Dec 2 2009

Related:

• Animation of influenza replication
• The Biology of Influenza
• Hungry for hosts – gene flow in influenza virus

Among the negative RNA viruses, ambisense RNA viruses occupy a distinct niche. Ambisense viruses contain at least one ambisense RNA segment, i.e. an RNA that is in part of positive and in part of negative polarity. Because of this unique gene organization, one might expect ambisense RNA viruses to borrow expression strategies from both positive and negative RNA viruses. However, they have little in common with positive RNA viruses, but possess many features of negative RNA viruses. Transcription and/or replication of their RNAs appear generally to be coupled to translation. Such coupling might be important to ensure temporal control of gene expression, allowing the two genes of an ambisense RNA segment to be differently regulated. Ambisense viruses can infect one host asymptomatically and in certain cases, they can lethally infect two hosts of a different kingdom. A possible model to explain the differential behavior of a given virus in different hosts could be that perturbation of the translation machinery would lead to differences in the severity of symptoms.

The ambisense coding strategy is an unusual way of encoding genes that presumably allows the virus to temporally control expression of the viral proteins, in particular if coupling of translation to transcription enhances the level of vc-encoded versus v-encoded protein expression. In any event, translation itself and/or translational control appear to play an important role in regulation of gene expression of ambisense viruses. Ambisense viruses have two hosts in which they can replicate. In their vector or reservoir host, infection is usually asymptomatic. However, in another host, multiplication of the virus can be lethal. Replication/transcription experiments in different host cell types would be helpful to shed further light on the differences observed in different hosts. At present, there are many complementary ways to study ambisense virus replication/ transcription such as cell culture, in vitro assays and reverse genetics systems. Since ambisense viruses are the meeting point of different viral families and are able to replicate in different hosts whether plants or animals and have different behaviors depending on the host, it would be particularly important to better understand the complex replicative cycle of ambisense viruses, in order to find the means to alleviate the lethal aspects of these pathogens.

Expression strategies of ambisense viruses. Virus Research 93: 141-150, 2003. doi: 10.1016/S0168-1702(03)00094-7

Related:

• Negative sense RNA viruses
• MicrobiologyBytes: Arenaviruses
• MicrobiologyBytes: Bunyaviruses

Retroviruses package two copies of full-length RNA in one virus particle (virion). One of the consequences of packaging two RNAs is frequent recombination during DNA synthesis when reverse transcriptase uses parts of both RNAs as templates. Although frequent recombination can occur during DNA synthesis of all virions, a genotypically different recombinant can only be generated from virions that package two different RNAs (heterozygous virions). High genetic diversity of HIV-1 presents a difficult barrier for drug treatment and vaccine development.

Using a recombination assay, researchers have shown that RNA molecules derived from two similar HIV-1 proviruses can randomly assort and be efficiently copackaged into virions. However, heterozygous virions are formed less efficiently when the two proviruses contain variations in their dimerization initiation signal (DIS). Located at the loop of stem-loop 1 of the 5′ untranslated region, the DIS is a 6-nt palindromic sequence that forms the initial interaction between the two HIV-1 RNAs. The Gag polyproteins of HIV-1 interact with, and specifically package, the viral RNA to generate infectious viruses. We have previously examined whether RNA dimerization occurs prior to virus assembly using HIV-1 variants with DIS mutations that abolish their palindromic nature (for example, from GCGCGC to GGGGGG) but can form perfect base pairs with the DIS of a partner virus (such as a virus with CCCCCC at the DIS). In the coinfected cells if dimeric RNAs are packaged, then the GGGGGG viral RNA would preferentially pair with CCCCCC viral RNA, and an increase in the formation of heterozygous virions would be observed. In contrast, if two monomeric RNAs are packaged, then there would not be an increase in heterozygous viruses. Results reveal that most of the virions from coinfected cells were heterozygous, indicating that copackaged RNA partner selection, i.e. dimerization, occurs prior to the packaging of virion RNA.

HIV-1 full-length RNAs serve at least two functions: as a template for Gag/Gag-Pol translation, and as genetic material packaged in the virion. Many cellular factors ensure the correct macromolecular trafficking between nucleus and cytoplasm; specifically, mechanisms exist to prevent the export of intron-containing transcripts, such as the full-length HIV-1 RNA. Most cellular mRNAs are fully spliced before export and many are believed to exit the nucleus via the NXF1-dependent pathway. However, many proteins and some RNAs use an alternative, CRM-1-dependent pathway to migrate out of the nucleus. The extent to which these two pathways are linked or overlap is currently unknown, and the reason for their differential use is subject to speculation.

Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, scientists sought to identify the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, they were able to demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, they showed that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, they demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.

Probing the HIV-1 Genomic RNA Trafficking Pathway and Dimerization by Genetic Recombination and Single Virion Analyses. 2009 PLoS Pathog 5(10): e1000627 doi:10.1371/journal.ppat.1000627

Related:

• The shape of HIV RNA
• Studying the structure of HIV

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease that can lead to appreciable morbidity including cirrhosis and hepatocellular carcinoma (HCC). HCV can be classified into several genotypes based on variations in the nucleotide sequence of its genome. The infecting HCV type has been shown to be clinically important because it predicts response to antiviral therapy, with infection by type 1 being associated with the most resistance to treatment. There is no consensus as to whether differences in the clinical and histological severity of liver disease can be explained by differences in the infecting type. Several studies suggest genotype 1b to be associated with more severe disease, but most have found little or no influence of genotype on disease progression. Similar ambiguity surrounds the study of the role of HCV genotypes in spontaneous viral clearance. Some studies have found no association between the viral type and the spontaneous clearance of HCV RNA, with host factors like sex seeming to be more important, while other studies have suggested that infection by genotype 1 or 1b might be less likely to clear spontaneously when compared with infection by other genotypes.
The aim of this study was to investigate whether the HCV type might influence the clinical outcome of infection. Study serum samples were assembled from 749 individuals enrolled into the UK HCV National Register from which data on clinical outcomes were determined. The prevalence of HCV type 1 among those who cleared infection was 69% and among those who remained HCV RNA positive was 51%: type 1 infections were more likely to be HCV RNA negative than non-1 types. Type 1 infections were also more likely to be associated with histological stage scores above the median when compared with non-1 types. In conclusion, HCV type 1 infection was more often HCV RNA negative, suggesting that spontaneous clearance may occur more commonly with this type. Among the RNA-positive infections, type 1 infection may be more aggressive than types 2/3.

Does the clinical outcome of hepatitis C infection vary with the infecting hepatitis C virus type?
J Viral Hepat. 2007 14: 213-220