The packaging signal of influenza viral RNA molecules.

The packaging signal present in influenza viral RNA molecules is shown not to constitute a separate structural element, but to reside within the 5'-bulged promoter structure, as caused by the central unpaired residue A10 in its 5' branch. Upon insertion of two uridine residues in the 3' branch opposite A10, the minus-strand viral RNA (vRNA) promoter is converted into a 3'-bulged structure, whereas the plus-strand cRNA promoter instead adopts the 5'-bulged conformation. In this promoter variant it is exclusively the cRNA that is found packaged in the progeny virions. Upon insertion of only a single uridine nucleotide opposite 5'A10, the two debulged structures of the vRNA and cRNA promoters are rendered identical, and both vRNA and cRNA molecules are packaged indiscriminately, in a 1:1 ratio, but at lower rates. We propose that the binding interactions of viral polymerase with either of the two differently bulged vRNA and cRNA promoter structures result in two different conformations of the enzyme protein. Only the 5' bulged RNA-associated polymerase conformation appears to be recognized for nuclear export, which depends on nuclear matrix protein M1 and nonstructural protein NS2. And the respective wild-type vRNP- or insertion mutant cRNP complex is observed to enter the cytoplasm and hence is included in the viral encapsidation process, which takes place at the plasma membrane.     

Strongly correlated electrostatics of viral genome packaging

The problem of viral packaging (condensation) and ejection from viral capsid in the presence of multivalent counterions is considered. Experiments show divalent counterions strongly influence the amount of DNA ejected from bacteriophage. In this paper, the strong electrostatic interactions between DNA molecules in the presence of multivalent counterions is investigated. It is shown that experiment results agree reasonably well with the phenomenon of DNA reentrant condensation. This phenomenon is known to cause DNA condensation in the presence of tri- or tetra-valent counterions. For divalent counterions, the viral capsid confinement strongly suppresses DNA configurational entropy, therefore the correlation between divalent counterions is strongly enhanced causing similar effect. Computational studies also agree well with theoretical calculations.     

Energy-independent helicase activity of a viral genome packaging motor

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.     

Evidence for segment-nonspecific packaging of the influenza a virus genome

The influenza A virus genome is composed of eight negative-sense RNA segments (called vRNAs), all of which must be packaged to produce an infectious virion. It is not clear whether individual vRNAs are packaged specifically or at random, however, and the total vRNA capacity of the virion is unknown. We have created modified forms of the viral nucleoprotein (NP), neuraminidase (NA), and nonstructural (NS) vRNAs that encode green or yellow fluorescent proteins and studied the efficiency with which these are packaged by using a plasmid-based influenza A virus assembly system. Packaging was assessed precisely and quantitatively by scoring transduction of the fluorescent markers in a single-round infectivity assay with a flow cytometer. We found that, under conditions in which virions are limiting, pairs of alternatively tagged vRNAs compete for packaging but do so in a nonspecific manner. Reporters representing different vRNAs were not packaged additively, as would be expected under specific packaging, but instead appeared to compete for a common niche in the virion. Moreover, 3 to 5% of transduction-competent viruses were found to incorporate two alternative reporters, regardless of whether those reporters represented the same or different vRNAs - a finding compatible with random, but not with specific, packaging. Probabilistic estimates suggest that in order to achieve this level of dual transduction by chance alone, each influenza A virus virion must package an average of 9 to 11 vRNAs.     

Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution

Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.     

Codon conservation in the influenza A virus genome defines RNA packaging signals

Genome segmentation facilitates reassortment and rapid evolution of influenza A virus. However, segmentation complicates particle assembly as virions must contain all eight vRNA species to be infectious. Specific packaging signals exist that extend into the coding regions of most if not all segments, but these RNA motifs are poorly defined. We measured codon variability in a large dataset of sequences to identify areas of low nucleotide sequence variation independent of amino acid conservation in each segment. Most clusters of codons showing very little synonymous variation were located at segment termini, consistent with previous experimental data mapping packaging signals. Certain internal regions of conservation, most notably in the PA gene, may however signify previously unidentified functions in the virus genome. To experimentally test the bioinformatics analysis, we introduced synonymous mutations into conserved codons within known packaging signals and measured incorporation of the mutant segment into virus particles. Surprisingly, in most cases, single nucleotide changes dramatically reduced segment packaging. Thus our analysis identifies cis-acting sequences in the influenza virus genome at the nucleotide level. Furthermore, we propose that strain-specific differences exist in certain packaging signals, most notably the haemagglutinin gene; this finding has major implications for the evolution of pandemic viruses.     

The influenza A virus genome packaging network — complex,flexible and yet unsolved

The genome of influenza A virus (IAV) consists of eight unique viral RNA segments. This genome organization allows genetic reassortment between co-infecting IAV strains, whereby new IAVs with altered genome segment compositions emerge. While it is known that reassortment events can create pandemic IAVs, it remains impossible to anticipate reassortment outcomes with pandemic prospects. Recent research indicates that reassortment is promoted by a viral genome packaging mechanism that delivers the eight genome segments as a supramolecular complex into the virus particle. This finding holds promise of predicting pandemic IAVs by understanding the intermolecular interactions governing this genome packaging mechanism. Here, we critically review the prevailing mechanistic model postulating that IAV genome packaging is orchestrated by a network of intersegmental RNA–RNA interactions. Although we find supporting evidence, including segment-specific packaging signals and experimentally proposed RNA–RNA interaction networks, this mechanistic model remains debatable due to a current shortage of functionally validated intersegmental RNA–RNA interactions. We speculate that identifying such functional intersegmental RNA–RNA contacts might be hampered by limitations of the utilized probing techniques and the inherent complexity of the genome packaging mechanism. Nevertheless, we anticipate that improved probing strategies combined with a mutagenesis-based validation could facilitate their discovery.     

Insights into specific DNA recognition during the assembly of a viral genome packaging machine

Terminase enzymes mediate genome "packaging" during the reproduction of DNA viruses. In lambda, the gpNu1 subunit guides site-specific assembly of terminase onto DNA. The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy. Its fold contains a unique winged helix-turn-helix (wHTH) motif within a novel scaffold. Surprisingly, a predicted P loop ATP binding motif is in fact the wing of the DNA binding motif. Structural and genetic analysis has identified determinants of DNA recognition specificity within the wHTH motif and the DNA recognition sequence. The structure reveals an unexpected DNA binding mode and provides a mechanistic basis for the concerted action of gpNu1 and Escherichia coli integration host factor during assembly of the packaging machinery.     

Replication-coupled and host factor-mediated encapsidation of the influenza virus genome by viral nucleoprotein

The influenza virus RNA-dependent RNA polymerase is capable of initiating replication but mainly catalyzes abortive RNA synthesis in the absence of viral and host regulatory factors. Previously, we reported that IREF-1/minichromosome maintenance (MCM) complex stimulates a de novo initiated replication reaction by stabilizing an initiated replication complex through scaffolding between the viral polymerase and nascent cRNA to which MCM binds. In addition, several lines of genetic and biochemical evidence suggest that viral nucleoprotein (NP) is involved in successful replication. Here, using cell-free systems, we have shown the precise stimulatory mechanism of virus genome replication by NP. Stepwise cell-free replication reactions revealed that exogenously added NP free of RNA activates the viral polymerase during promoter escape while it is incapable of encapsidating the nascent cRNA. However, we found that a previously identified cellular protein, RAF-2p48/NPI-5/UAP56, facilitates replication reaction-coupled encapsidation as an NP molecular chaperone. These findings demonstrate that replication of the virus genome is followed by its encapsidation by NP in collaboration with its chaperone.     

The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging

The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.     

Atomistic basis of force generation,translocation, and coordination in a viral genome packaging motor

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.     

The segments of influenza viral mRNA.

Influenza viral mRNA, i.e., complementary RNA (cRNA), isolated from infected cells , was resolved into six different species by electrophoresis in 2.1% acrylamide gels containing 6 M urea. The cRNA''s were grouped into three size classes: L (large), M (medium-size), and S (small). Similarly, when gels were sliced for analysis, the virion RNA (vRNA) also distributed into six peaks because the three largest vRNA segments were closely spaced and were resolved only when the gels were autoradiographed or stained. Because of their attached polyadenylic acid [poly(A)]sequences, the cRNA segments migrated more slowly than did the corresponding vRNA segments during gel electrophoresis. After removal of the poly(A) by RNase H, the cRNA and vRNA segments comigrated, indicating that they were approximately the same size. One of the cRNA segments, S2, was shown by annealing to contain the genetic information in the vRNA segment with which it comigrated, strongly suggesting that each cRNA segment was transcribed from the vRNA segment of the same size. In contrast to the vRNA segments, which when isolated from virions were present in approximately 1:1 molar ratios, the segments of the isolated cRNA were present in unequal amounts, with the segments M2 and S2 predominating, suggesting that different amounts of the cRNA segments were synthesized in the infected cell. The predominant cRNA segments, M2 and S2, and also the S1 segment, were active as mRNA''s in wheat germ extracts. The M2 cRNA was the mRNA for the nucleocapsid protein; S1 for the membrane protein; and S2 for the nonstructural protein NS1.     

A型流感病毒的基因组包装

A型流行性感冒病毒的负链RNA基因组由编码病毒中12个蛋白质的八个节段组成。在病毒组装的最后阶段，病毒体从细胞顶端胞浆膜突出时将这些基因组的病毒体（v）RNAs吸收进其中。基因组分段赋予了流感病毒进化的优势，但也提出了问题，在病毒体组装时需要八个节段每一个的至少一个复制本以产生完全有传染性的病毒颗粒。历史上一直存在争论：一方赞同确保足额的基因组合并的特异性包装机制；另一方赞同基因组节段被随机选择而不是以充足数量被包装以确保能自行产生合理比例病毒体的替代模式。近年来人们对该问题已达成一致意见：大多数病毒体仅包含八个节段，特异性机制为选择每个vRNA的某一复制本的确发挥了作用。本综述总结了得出这一结论所做的工作，叙述了在识别特异性包装信号方面最新的进展，讨论了这些RNA元素运转的可能机制。     

Chromosome condensation: packaging the genome

The packaging of centimetre long DNA molecules into compact metaphase chromosomes is essential for genome segregation in anaphase. The chromosomal condensin complex plays a crucial part in this packaging, and important new insight into condensin action in vitro and in vivo has recently been gained.     

The influenza A virus PB2, PA, NP, and M segments play a pivotal role during genome packaging

The genomes of influenza A viruses consist of eight negative-strand RNA segments. Recent studies suggest that influenza viruses are able to specifically package their segmented genomes into the progeny virions. Segment-specific packaging signals of influenza virus RNAs (vRNAs) are located in the 5' and 3' noncoding regions, as well as in the terminal regions, of the open reading frames. How these packaging signals function during genome packaging remains unclear. Previously, we generated a 7-segmented virus in which the hemagglutinin (HA) and neuraminidase (NA) segments of the influenza A/Puerto Rico/8/34 virus were replaced by a chimeric influenza C virus hemagglutinin/esterase/fusion (HEF) segment carrying the HA packaging sequences. The robust growth of the HEF virus suggested that the NA segment is not required for the packaging of other segments. In this study, in order to determine the roles of the other seven segments during influenza A virus genome assembly, we continued to use this HEF virus as a tool and analyzed the effects of replacing the packaging sequences of other segments with those of the NA segment. Our results showed that deleting the packaging signals of the PB1, HA, or NS segment had no effect on the growth of the HEF virus, while growth was greatly impaired when the packaging sequence of the PB2, PA, nucleoprotein (NP), or matrix (M) segment was removed. These results indicate that the PB2, PA, NP, and M segments play a more important role than the remaining four vRNAs during the genome-packaging process.     

SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2' hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem-loops, extensive long-range interactions (LRIs) and a small, palindromic stem-loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Our analyses of wild-type (wt) and mutant RNAs suggest that although the four conserved stem-loops are static structures, the 5' and 3' regions previously shown to form LRI also adopt an alternative, yet similarly conserved conformation, in which the putative DIS is occluded, and which may thus favour translational and splicing functions over encapsidation. SHAPE and in vitro dimerization assays were used to examine SL5 mutants. Dimerization contacts appear to be made between palindromic loop sequences in SL5. As this stem-loop is located within the gag ORF, recognition of a dimeric RNA provides a possible mechanism for the specific packaging of genomic over spliced viral RNAs.