Human Cytomegalovirus Inhibitor AL18 Also Possesses Activity against Influenza A and B Viruses

AL18, an inhibitor of human cytomegalovirus DNA polymerase, was serendipitously found to also block the interaction between the PB1 and PA polymerase subunits of influenza A virus. Furthermore, AL18 effectively inhibited influenza A virus polymerase activity and the overall replication of influenza A and B viruses. A molecular model to explain the binding of AL18 to both cytomegalovirus and influenza targets is proposed. Thus, AL18 represents an interesting lead for the development of new antivirals

Evolutionary Conservation of the PA-X Open Reading Frame in Segment 3 of Influenza A Virus

PA-X is a fusion protein of influenza A virus encoded in part from a +1 frameshifted X open reading frame (X-ORF) in segment 3. We show that the X-ORFs of diverse influenza A viruses can be divided into two groups that differ in selection pressure and likely function, reflected in the presence of an internal stop codon and a change in synonymous diversity. Notably, truncated forms of PA-X evolved convergently in swine and dogs, suggesting a strong species-specific effect

Compositional biases in RNA viruses::causes, consequences and applications

If each of the four nucleotides were represented equally in the genomes of viruses and the hosts they infect, each base would occur at a frequency of 25%. However, this is not observed in nature. Similarly, the order of nucleotides is not random (e.g., in the human genome, guanine follows cytosine at a frequency of ~0.0125, or a quarter the number of times predicted by random representation). Codon usage and codon order are also nonrandom. Furthermore, nucleotide and codon biases vary between species. Such biases have various drivers, including cellular proteins that recognize specific patterns in nucleic acids, that once triggered, induce mutations or invoke intrinsic or innate immune responses. In this review we examine the types of compositional biases identified in viral genomes and current understanding of the evolutionary mechanisms underpinning these trends. Finally, we consider the potential for large scale synonymous recoding strategies to engineer RNA virus vaccines, including those with pandemic potential, such as influenza A virus and Severe Acute Respiratory Syndrome Coronavirus Virus 2. This article is categorized under: RNA in Disease and Development > RNA in Disease. RNA Evolution and Genomics > Computational Analyses of RNA. RNA Interactions with Proteins and Other Molecules > Protein‐RNA Recognition

Permissive and Restricted Virus Infection of Murine Embryonic Stem Cells

Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence

Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle

The genetics of virus particle shape in equine influenza A virus

Background Many human strains of influenza A virus produce highly pleomorphic virus particles that at the extremes can be approximated as either spheres of around 100 nm diameter or filaments of similar cross-section but elongated to lengths of many microns. The role filamentous virions play in the virus life cycle remains enigmatic. Objectives/Methods Here, we set out to define the morphology and genetics of virus particle shape in equine influenza A virus, using reverse genetics and microscopy of infected cells. Results and Conclusions The majority of H3N8 strains tested were found to produce filamentous virions, as did the prototype H7N7 A/eq/Prague/56 strain. The exception was the prototype H3N8 isolate, A/eq/Miami/63. Reassortment of equine influenza virus M genes from filamentous and non-filamentous strains into the non-filamentous human virus A/PR/8/34 confirmed that segment 7 is a major determinant of particle shape. Sequence analysis identified three M1 amino acid polymorphisms plausibly associated with determining virion morphology, and the introduction of these changes into viruses confirmed the importance of two: S85N and N231D. However, while either change alone affected filament production, the greatest effect was seen when the polymorphisms were introduced in conjunction. Thus, influenza A viruses from equine hosts also produce filamentous virions, and the major genetic determinants are set by the M1 protein. However, the precise sequence determinants are different to those previously identified in human or porcine viruses

Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein

The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33 ts56 and A/FPV/Rostock/34/Giessen tsG81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30 degrees C. This suggests that the RNA-binding activity of NP is required for replicative transcription

Activation of influenza virus RNA polymerase by the 5' and 3' terminal duplex of genomic RNA

The current model for influenza virus mRNA transcription involves the sequential interaction of the viral polymerase with the 5'- and 3'-ends of vRNA, with each RNA-protein interaction triggering a polymerase function necessary for cap-primed transcription. Here we show that the order in which this ternary complex is assembled is in fact important. Polymerase bound simultaneously to a pre-annealed duplex of the 5'- and 3'-ends of vRNA had greatly increased levels of primer binding and endonuclease activities compared to a sequentially assembled complex. Increased primer binding was due to the activation of a high affinity binding site with a preference for primer length RNAs. This correlated with enhanced levels of cap-primed transcription. Polymerase that was bound initially to just 5' vRNA had low primer binding activity, but was endonucleolytically active. Neither activity was significantly increased by the subsequent addition of 3' vRNA, and this sequentially assembled complex had correspondingly low mRNA transcription activity. Nevertheless, both routes of assembly led to complexes that were highly competent for dinucleotide ApG-primed transcription. Therefore, polymerase complexes assembled on pre-annealed 5' and 3' terminal viral RNA sequences have distinct properties from those assembled by sequential loading of polymerase onto the 5'-end followed by the 3'-end. This suggests a mechanism by which the virus couples transcription initiation and termination during mRNA transcription

Functional domains of the influenza A virus PB2 protein:identification of NP- and PB1-binding sites

Influenza virus genomic RNA segments are packaged into ribonucleoprotein (RNP) structures by the PB1, PB2, and PA subunits of an RNA polymerase and a single-strand RNA-binding nucleoprotein (NP). Assembly and function of these ribonucleoproteins depend on a complex set of protein-protein and protein-RNA interactions. Here, we identify new functional domains of PB2. We show that PB2 contains two regions that bind NP and also identify a novel PB1 binding site. The regions of PB2 responsible for binding NP and PB1 show considerable overlap, and binding of NP to the PB2 fragments could be outcompeted by PB1. The binding domains of PB2 acted as trans-dominant inhibitors of viral gene expression, and consistent with the in vitro binding data, their inhibitory activity depended on the concentration of wild-type PB2, NP, and PB1. This provides evidence for functionally significant and potentially regulatory interactions between PB2 and NP