Juan Ortín

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance-a review

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.

Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model.

Avian influenza A H5N1 viruses continue to spread globally among birds, resulting in occasional transmission of virus from infected poultry to humans. Probable human-to-human transmission has been documented rarely, but H5N1 viruses have not yet acquired the ability to transmit efficiently among humans, an essential property of a pandemic virus. The pandemics of 1957 and 1968 were caused by avian–human reassortant influenza viruses that had acquired human virus-like receptor binding properties. However, the relative contribution of human internal protein genes or other molecular changes to the efficient transmission of influenza viruses among humans remains poorly understood. Here, we report on a comparative ferret model that parallels the efficient transmission of H3N2 human viruses and the poor transmission of H5N1 avian viruses in humans. In this model, an H3N2 reassortant virus with avian virus internal protein genes exhibited efficient replication but inefficient transmission, whereas H5N1 reassortant viruses with four or six human virus internal protein genes exhibited reduced replication and no transmission. These findings indicate that the human virus H3N2 surface protein genes alone did not confer efficient transmissibility and that acquisition of human virus internal protein genes alone was insufficient for this 1997 H5N1 virus to develop pandemic capabilities, even after serial passages in a mammalian host. These results highlight the complexity of the genetic basis of influenza virus transmissibility and suggest that H5N1 viruses may require further adaptation to acquire this essential pandemic trait.

Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport.

The influenza virus RNA segment 8 encodes two proteins, NS1 and NS2, by differential splicing. The collinear transcript acts as mRNA for NS1 protein, while the spliced mRNA encodes NS2 protein. The splicing of NS1 mRNA was studied in cells transfected with a recombinant plasmid that has the cDNA of RNA segment 8 cloned under the SV40 late promoter and polyadenylation signals. As described for influenza virus‐infected cells, NS1 mRNA was poorly spliced to yield NS2 mRNA. However, inactivation of the NS1 gene, but not the NS2 gene, led to a substantial increase in the splicing efficiency, as shown by the relative accumulations of NS1 and NS2 mRNAs. This effect was not specific for NS1 mRNA, since the splicing of the endogenous SV40 early transcript was altered in such a way that t‐Ag mRNA was almost eliminated. These changes in the splicing pattern coincided with a strong inhibition of the mRNA nucleocytoplasmic transport. Both NS1 and NS2 mRNAs were retained in the nucleus of cells expressing NS1 protein, but no effect was observed when only NS2 protein was expressed. Furthermore, other mRNAs tested, such as T‐Ag mRNA and the non‐spliceable nucleoprotein transcript, were also retained in the nucleus upon expression of NS1 protein, suggesting that it induced a generalized block of mRNA export from the nucleus.

Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture

The genetic heterogeneity generated upon passage of foot-and-mouth disease virus (FMDV) in cell culture has been evaluated by T1-oligonucleotide fingerprinting of genomic RNA. Plaque-purified FMDV O-S7 and C-S8 were propagated by serial low multiplicity infections of BHK-21 (c-13) or IBRS-2 (c-26) cells. In independent parallel passage of the same virus, different oligonucleotide variations were fixed in the RNAs. T1-oligonucleotide fingerprinting of RNA from 34 individual viral clones derived from two passaged populations shows an extensive heterogeneity, with some mutations present in only one of the cloned genomes analyzed. Some FMDV variants are phenotypically distinct in that they yield increased progeny in infections of cell monolayers. From the number of variant sequences it can be estimated that each infectious RNA in the population differs in two to eight mutations from the average parental sequence. Thus, passaged FMDV populations consist of a pool of variants, an observation previously made with phage Q beta (E. Domingo, D. Sabo, T. Taniguchi, and C. Weissmann, Cell 13, 735-744, 1978). The FMDV genome must be described as a fluctuating distribution of sequences due to its high mutability. This may be the basis of the extensive genetic and antigenic diversity of this virus in nature.

Eukaryotic Translation Initiation Factor 4GI Is a Cellular Target for NS1 Protein, a Translational Activator of Influenza Virus

ABSTRACT Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5′ untranslated region (5′ UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

The structure of a biologically active influenza virus ribonucleoprotein complex

The influenza viruses contain a segmented, single-stranded RNA genome of negative polarity. Each RNA segment is encapsidated by the nucleoprotein and the polymerase complex into ribonucleoprotein particles (RNPs), which are responsible for virus transcription and replication. Despite their importance, information about the structure of these RNPs is scarce. We have determined the three-dimensional structure of a biologically active recombinant RNP by cryo-electron microscopy. The structure shows a nonameric nucleoprotein ring (at 12 Å resolution) with two monomers connected to the polymerase complex (at 18 Å resolution). Docking the atomic structures of the nucleoprotein and polymerase domains, as well as mutational analyses, has allowed us to define the interactions between the functional elements of the RNP and to propose the location of the viral RNA. Our results provide the first model for a functional negative-stranded RNA virus ribonucleoprotein complex. The structure reported here will serve as a framework to generate a quasi-atomic model of the molecular machine responsible for viral RNA synthesis and to test new models for virus RNA replication and transcription.

The Structure of Native Influenza Virion Ribonucleoproteins

Influenza Revealed Influenza virus, a single-stranded RNA virus, is responsible for substantial morbidity and mortality worldwide. The influenza ribonucleoprotein (RNP) complex, which carries out viral replication and transcription, is central to the virus life-cycle and to viral host adaptation (see the Perspective by Tao and Zheng). Structural characterization of the viral RNP has been challenging, but Moeller et al. (p. 1631, published online 22 November) and Arranz et al. (p. 1634, published online 22 November) now report the structure and assembly of this complex, using cryo-electron microscopy and negative-stain electron microscopy. The structures reveal how the viral polymerase, RNA genome, and nucleoprotein interact in the RNP providing insight into mechanisms for influenza genome replication and transcription. Electron microscopic analysis of a purified RNA-protein complex links its structure to the influenza life cycle. The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics, which constitute a major public-health issue. The segmented negative-stranded RNAs are associated with the polymerase complex and nucleoprotein (NP), forming ribonucleoproteins (RNPs), which are responsible for virus transcription and replication. We describe the structure of native RNPs derived from virions. They show a double-helical conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase complex at the other end. This structure will be relevant for unraveling the mechanisms of nuclear import of parental virus RNPs, their transcription and replication, and the encapsidation of progeny RNPs into virions.

A Human Sequence Homologue of Staufen Is an RNA-Binding Protein That Is Associated with Polysomes and Localizes to the Rough Endoplasmic Reticulum

ABSTRACT In the course of a two-hybrid screen with the NS1 protein of influenza virus, a human clone capable of coding for a protein with high homology to the Staufen protein from Drosophila melanogaster (dmStaufen) was identified. With these sequences used as a probe, cDNAs were isolated from a λ cDNA library. The encoded protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding. A recombinant protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide. It showed dsRNA binding activity in vitro, with an apparentKd of 10−9 M. Using a specific antibody, we detected in human cells a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa. The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicated that hStaufen-like protein associates with polysomes. These results are discussed in relation to the possible functions of the protein.

Efficient transformation of mammalian cells with constructs containing a puromycin-resistance marker

Recombinant plasmids have been obtained that lead to the accumulation of five- to ten-fold more puromycin-N-acetyl-transferase (PAC) mRNA and two- to three-fold more PAC activity than the already described plasmid pSV2pac [Vara et al., Nucl. Acids Res. 14 (1986) 4117-4124]. When these optimized recombinants were used for stable transformation to puromycin resistance, efficiencies up to 1 x 10(-2) were obtained, indicating that these pac-containing recombinants may be very useful dominant selectable markers for gene transfer in mammalian cells.

The influenza virus RNA synthesis machine: Advances in its structure and function

The influenza A viruses are the causative agents of respiratory disease that occurs as yearly epidemics and occasional pandemics. These viruses are endemic in wild avian species and can sometimes break the species barrier to infect and generate new virus lineages in humans. The influenza A virus genome consists of eight single-stranded, negative-polarity RNAs that form ribonucleoprotein complexes by association to the RNA polymerase and the nucleoprotein. In this review we focus on the structure of this RNA-synthesis machines and the included RNA polymerase, and on the mechanisms by which they express their genetic information as mRNAs and generate progeny ribonucleoproteins that will become incorporated into new infectious virions. New structural, biochemical and genetic data are rapidly accumulating in this very active area of research. We discuss these results and attempt to integrate the information into structural and functional models that may help the design of new experiments and further our knowledge on virus RNA replication and gene expression. This interplay between structural and functional data will eventually provide new targets for controlled attenuation or antiviral therapy