Defective Influenza Viral Ribonucleoproteins Cause Interference

Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.     

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.     

Characterization of influenza virus NS1 protein by using a novel helper-virus-free reverse genetic system

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants, dl12 and N110, using this protocol. The dl12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39 degrees C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.     

Introduction of a temperature-sensitive phenotype into influenza A/WSN/33 virus by altering the basic amino acid domain of influenza virus matrix protein

Our previous studies with influenza A viruses indicated that the association of M1 with viral RNA and nucleoprotein (NP) is required for the efficient formation of helical ribonucleoprotein (RNP) and for the nuclear export of RNPs. RNA-binding domains of M1 map to the following two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105. Altering the zinc finger motif of M1 reduces viral growth slightly. A substitution of Ser for Arg at either position 101 or position 105 of the RKLKR domain partially reduces the nuclear export of RNP and viral replication. To further understand the role of the zinc finger motif and the RKLKR domain in viral assembly and replication, we introduced multiple mutations by using reverse genetics to modify these regions of the M gene of influenza virus A/WSN/33. Of multiple mutants analyzed, a double mutant, R101S-R105S, of RKLKR resulted in a temperature-sensitive phenotype. The R101S-R105S double mutant had a greatly reduced ratio of M1 to NP in viral particles and a weaker binding of M1 to RNPs. These results suggest that mutations can be introduced into the RKLKR domain to control viral replication.     

The Putative Polymerase Sequence of Infectious Salmon Anemia Virus Suggests a New Genus within the Orthomyxoviridae

The infectious salmon anemia virus (ISAV) is an orthomyxovirus-like virus infecting teleosts. The disease caused by this virus has had major economic consequences for the Atlantic salmon farming industry in Norway, Canada, and Scotland. In this work, we report the cloning and sequencing of an ISAV-specific cDNA comprising 2,245 bp with an open reading frame coding for a predicted protein with a calculated molecular weight of 80.5 kDa. The putative protein sequence shows the core polymerase motifs characteristic of all viral RNA-dependent RNA polymerases. Comparison of the conserved motifs with the corresponding regions of other segmented negative-stranded RNA viruses shows a closer relationship with members of the Orthomyxoviridae than with viruses in other families. The putative ISAV polymerase protein (PB1) has a length of 708 amino acids, a charge of +22 at neutral pH, and a pI of 9.9, which are consistent with the properties of the PB1 proteins of other members of the family. Calculations of the distances between the different PB1 proteins indicate that the ISAV is distantly related to the other members of the family but more closely related to the influenza viruses than to the Thogoto viruses. Based on these and previously published results, we propose that the ISAV comprises a new, fifth genus in the Orthomyxoviridae.     

The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes

The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain.     

Reconstitution of influenza virus RNA-nucleoprotein complexes structurally resembling native viral ribonucleoprotein cores

Reconstitution of influenza virus nucleoprotein (NP)-RNA complexes was performed with segment 8 RNA, which was synthesized in vitro from cDNA, and NP purified from virions. Under optimum conditions established using a filter binding assay and a gel retardation assay, NP was found to bind any RNA longer than 15 nucleotides. NP-RNA complexes formed at 30 degrees C are more resistant to high concentrations of NaCl than those formed at 0 degrees C. Treatment of NP with N-ethylmaleimide gave no effect on its RNA binding activity, whereas treatment with alkaline phosphatase enhanced its RNA binding activity. The newly developed "reverse-printing" method of RNase V1-treated complexes revealed that reconstituted NP-RNA complexes carry RNase V1-sensitive sites as do native ribonucleoprotein (RNP) cores (RNA polymerase-NP-RNA complexes), implying that RNA-NP complexes structurally similar to native RNP cores are reconstituted from isolated components.     

A Nine-Segment Influenza A Virus Carrying Subtype H1 and H3 Hemagglutinins

Influenza virus genomic RNAs possess segment-specific packaging signals that include both noncoding regions (NCRs) and adjacent terminal coding region sequences. Using reverse genetics, an A/Puerto Rico/8/34 (A/PR/8/34) virus was rescued that contained a modified PB1 gene such that the PB1 packaging sequences were exchanged for those of the neuraminidase (NA) gene segment. To accomplish this, the PB1 open reading frame, in which the terminal packaging signals were inactivated by serial synonymous mutations, was flanked by the NA segment-specific packaging sequences including the NCRs and the coding region packaging signals. Next, the ATGs located on the 3′ end of the NA packaging sequences of the resulting PB1 chimeric segment were mutated to allow for correct translation of the full-length PB1 protein. The virus containing this chimeric PB1 segment was viable and able to stably carry a ninth, green fluorescent protein (GFP), segment flanked by PB1 packaging signals. Utilizing this method, we successfully generated an influenza virus that contained the genes coding for both the H1 hemagglutinin (HA) from A/PR/8/34 and the H3 HA from A/Hong Kong/1/68 (A/HK/1/68); both subtypes of HA protein were also incorporated into the viral envelope. Immunization of mice with this recombinant virus conferred complete protection from lethal challenge with recombinant A/PR/8/34 virus and with X31 virus that expresses the A/HK/1/68 HA and NA. Using the described methodology, we show that a ninth segment can also be incorporated by manipulation of the PB2 or PA segment-specific packaging signals. This approach offers a means of generating a bivalent influenza virus vaccine.Influenza viruses possess segmented, negative-sense RNA genomes and belong to the family of Orthomyxoviridae. Three types of influenza viruses have been identified: A, B, and C (24). Based on the two surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), type A viruses are further divided into different subtypes; there are now 16 HA subtypes (H1 to H16) and 9 NA subtypes (N1 to N9) of influenza A viruses (24). Current influenza A viruses circulating in humans include the H1N1 and H3N2 subtypes.The genomes of influenza A and B viruses consist of eight RNAs, while C viruses have only seven segments. Influenza virus genomic RNAs associate with nucleoprotein (NP) and three viral polymerase subunits (PB2, PB1, and PA), to form the ribonucleoprotein (RNP) complexes within virions (24). Previous data indicated that each segment of the influenza A/WSN/33 (H1N1) virus possesses segment-specific RNA packaging signals that include both the 3′ and 5′ noncoding regions (NCRs), as well as coding sequences at the two ends of each open reading frame (ORF) (4, 5, 10, 11, 13, 15, 22, 23, 28; and see Fig. 47.23 in reference 24). In addition, an electron microscopy study showed that the wild-type influenza A virus contains exactly eight RNPs within the virions, with seven RNPs surrounding a central one (19). These results suggest that influenza virus genome packaging is a specific process, with each particle containing eight unique RNA segments. Additional evidence supporting a specific packaging theory came from studies of defective interfering (DI) RNAs which contain internal deletions in the coding sequences. These short RNAs can be incorporated into the virus particles despite the fact that they do not encode full-length functional proteins. The finding that incorporation of DI RNAs interferes with the parent full-length RNAs in a segment-specific manner (1, 16, 17) also suggests that influenza virus genome packaging is a specific process.However, there are also data arguing that influenza virus RNA packaging can be nonspecific. First, studies showed that the two different RNA segments of influenza virus can be engineered to share the same set of 3′ and 5′ NCRs, which are important components of the influenza virus RNA packaging signals (18, 31). In addition, under specific circumstances, influenza virus is able to contain nine RNA segments, in which two of them share identical NCRs and partially identical coding region sequences (2, 29). Titrations of the nine-segment virus revealed a linear relationship between dilutions and plaque numbers, suggesting an influenza virus virion can incorporate more than eight segments (2).Herein, we describe a novel approach for the generation of nine-segment influenza viruses based on the manipulation of the segment-specific packaging signals. When the packaging sequences of the PB1 (or PB2 or PA) segment were replaced by those of the NA segment, influenza A/PR/8/34 virus was able to stably incorporate a ninth segment flanked by the PB1 (or PB2 or PA) packaging signals. Using this property, we successfully generated influenza viruses encoding two full-length HA glycoproteins: a subtype H1 A/PR/8/34 HA and a subtype H3 A/HK/1/68 HA. Immunization of mice with the virus carrying two HAs protected them from the lethal challenge with either A/PR/8/34 or X31 virus, the latter of which carries the HA and NA genes of A/HK/1/68. This approach can be used to construct live attenuated influenza vaccine viruses targeting two heterologous strains.     

Comparative aspects of infectious salmon anemia virus,an orthomyxovirus of fish,to influenza viruses

Infectious salmon anaemia (ISA) is a viral disease that was first recorded in 1984 in farmed Atlantic salmon. The infectious salmon anaemia virus (ISAV) is classified as the type species of the genus Isavirus in the Orthomyxoviridae family and is evolutionary remote to the influenza viruses. The genome consists of eight negative single-stranded RNA segments, and it utilises the same mechanisms as influenza viruses to enter and exit cells. Although a common ancestor of ISAV and other genera of Orthomyxoviruses could be dated back several millions of years, there are still many similarities between ISAV and the influenza viruses regarding morphology, replication cycles and interactions with their respective hosts.     

A seven-segmented influenza A virus expressing the influenza C virus glycoprotein HEF

Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.     

Cryotomography of Budding Influenza A Virus Reveals Filaments with Diverse Morphologies that Mostly Do Not Bear a Genome at Their Distal End

Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process.     

Isolation and Properties of an RNA Polymerase from Influenza Virus-Infected Cells

Structures with RNA polymerase activity were isolated from influenza virus-infected cells, and consisted of ribonucleoprotein (RNP) complexes, similar in morphology to the viral internal component or nucleocapsid. The isolation procedure involved fractionation of infected cells in a discontinuous sucrose gradient, in which enzyme activity was concentrated in a fraction of intermediate density which contains both smooth and rough cytoplasmic membranes. The RNPs with polymerase activity were further purified in a velocity gradient, after which the peak fractions showed a 35-fold purification of the polymerase activity when compared with cytoplasmic extracts. The NP polypeptide, which is the subunit of the virion RNP, was the only virus-specific polypeptide detected in these RNP structures.     

Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer

The genome of influenza A virus is organized into eight ribonucleoprotein complexes (RNPs), each containing one RNA polymerase complex. This RNA polymerase has also been found non-associated to RNPs and is possibly involved in distinct functions in the infection cycle. We have expressed the virus RNA polymerase complex by co-tranfection of the PB1, PB2 and PA genes in mammalian cells and the heterotrimer was purified by the TAP tag procedure. Its 3D structure was determined by electron microscopy and single-particle image processing. The model obtained resembles the structure previously reported for the polymerase complex associated to viral RNPs but appears to be in a more open conformation. Detailed model comparison indicated that specific areas of the complex show important conformational changes as compared to the structure for the RNP-associated polymerase, particularly in regions known to interact with the adjacent NP monomers in the RNP. Also, the PB2 subunit seems to undergo a substantial displacement as a result of the association of the polymerase to RNPs. The structural model presented suggests that a core conformation of the polymerase in solution exists but the interaction with other partners, such as proteins or RNA, will trigger distinct conformational changes to activate new functional properties.     

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV^901_09 and rISAVr^S6-NotI-HPR containing a NotI restriction site and rISAV^S6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 10⁵ PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.