Autophagy activation is required for influenza A virus-induced apoptosis and replication.

Autophagy and apoptosis are two major interconnected host cell responses to viral infection, including influenza A virus (IAV). Thus, delineating these events could facilitate the development of better treatment options and provide an effective anti-viral strategy for controlling IAV infection. We used A549 cells and mouse embryonic fibroblasts (MEF) to study the role of virus-induced autophagy and apoptosis, the cross-talk between both pathways, and their relation to IAV infection [ATCC strain A/Puerto Rico/8/34(H1N1) (hereafter; PR8)]. PR8-infected and mock-infected cells were analyzed by immunoblotting, immunofluorescence confocal microscopy, electron microscopy and flow cytometry (FACS). We found that PR8 infection simultaneously induced autophagy and apoptosis in A549 cells. Autophagy was associated with Bax and Bak activation, intrinsic caspase cleavage and subsequent PARP-1 and BID cleavage. Both Bax knockout (KO) and Bax/Bak double knockout MEFs displayed inhibition of virus-induced cytopathology and cell death and diminished virus-mediated caspase activation, suggesting that virus-induced apoptosis is Bax/Bak-dependent. Biochemical inhibition of autophagy induction with 3-methyladenine blocked both virus replication and apoptosis pathways. These effects were replicated using autophagy-refractory Atg3 KO and Atg5 KO cells. Taken together, our data indicate that PR8 infection simultaneously induces autophagy and Bax/caspase-dependent apoptosis, with autophagy playing a role to support PR8 replication, in part, by modulating virus-induced apoptosis.

Knockdown of specific host factors protects against influenza virus-induced cell death.

Cell death is a characteristic consequence of cellular infection by influenza virus. Mounting evidence indicates the critical involvement of host-mediated cellular death pathways in promoting efficient influenza virus replication. Furthermore, it appears that many signaling pathways, such as NF-κB, formerly suspected to solely promote cell survival, can also be manipulated to induce cell death. Current understanding of the cell death pathways involved in influenza virus-mediated cytopathology and in virus replication is limited. This study was designed to identify host genes that are required for influenza-induced cell death. The approach was to perform genome-wide lentiviral-mediated human gene silencing in A549 cells and determine which genes could be silenced to provide resistance to influenza-induced cell death. The assay proved to be highly reproducible with 138 genes being identified in independent screens. The results were independently validated using siRNA to each of these candidates. Graded protection was observed in this screen with the silencing of any of 19 genes, each providing > 85% protection. Three gene products, TNFSF13 (APRIL), TNFSF12-TNFSF13 (TWE-PRIL) and USP47, were selected because of the high levels of protection conferred by their silencing. Protein and mRNA silencing and protection from influenza-induced cell death was confirmed using multiple shRNA clones and siRNA, indicating the specificity of the effects. USP47 knockdown prevented proper viral entry into the host cell, whereas TNFSF12-13/TNFSF13 knockdown blocked a late stage in viral replication. This screening approach offers the means to identify a large number of potential candidates for the analysis of viral-induced cell death. These results may also have much broader applicability in defining regulatory mechanisms involved in cell survival.

Virus-triggered autophagy in viral hepatitis - possible novel strategies for drug development.

Autophagy is a very tightly regulated process that is important in many cellular processes including development, differentiation, survival and homoeostasis. The importance of this process has already been proven in numerous common diseases such as cancer and neurodegenerative disorders. Emerging data indicate that autophagy plays an important role in some liver diseases including liver injury induced by ischaemia reperfusion and alpha-1 antitrypsin Z allele-dependent liver disease. Autophagy may also occur in viral infection, and it may play a crucial role in antimicrobial host defence against pathogens, while supporting cellular homoeostasis processes. Here, the latest findings on the role of autophagy in viral hepatitis B and C infection, which are both serious health threats, will be reviewed.

Reovirus structure and morphogenesis.

Assembly of a mature infectious virion from component parts is one of the last steps in the replicative cycle of most viruses. Recent advances in delineating aspects of this process for the mammalian orthoreoviruses (MRV), nonenveloped viruses composed of a genome of ten segments of double-stranded RNA enclosed in two concentric icosahedral protein capsids, are discussed. Analyses of temperature-sensitive (ts) assembly-defective reovirus mutants have been used to better understand requirements for viral inclusion formation and capsid morphogenesis. Newly determined high-resolution structures of virtually all MRV proteins, combined with complete MRV genomic sequence information and elucidation of sequence lesions in ts mutants, is now providing a context for molecularly understanding interactions that promote, or abrogate, reovirus capsid assembly. Additional advances in understanding required signals for whole genome construction from sets of the ten individual genes, and in transcapsidation of subviral particles with engineered outer capsid proteins, provide additional molecular genetic understanding of reovirus protein structure-function and morphogenesis.

Generation and genetic characterization of avian reovirus temperature-sensitive mutants.

There currently is little known about the genetic and biological functions of avian reovirus (ARV), an atypical member of the family Reoviridae and the prototype of all nonenveloped viruses that induce syncytia formation. In this study, we created ARV temperature-sensitive (ts) mutants by chemical mutagenesis of ARV strain 138. We developed a novel efficiency of lysis (EOL) screening technique and used it and the classical efficiency of plating (EOP) assay to identify 17 ARV ts mutants. Pairwise mixed infections of these mutants and evaluation of recombinant progeny ts status led to their organization into seven recombination groups. This indicates that these new groups of mutants represent the majority of the ARV genome. To phenotypically characterize the ts mutants, progeny double-stranded RNA (dsRNA) produced at permissive and nonpermissive temperature was measured. Some mutants were capable of dsRNA synthesis at the restrictive temperature (RNA(+)), which indicates the effects of their ts lesions occur after RNA replication. Most mutants were RNA(-), which suggests their mutations affect stages in viral replication that precede progeny genome synthesis.

Avian reovirus major mu-class outer capsid protein influences efficiency of productive macrophage infection in a virus strain-specific manner.

We determined that the highly pathogenic avian reovirus strain 176 (ARV-176) possesses an enhanced ability to establish productive infections in HD-11 avian macrophages compared to avian fibroblasts. Conversely, the weakly pathogenic strain ARV-138 shows no such macrophagotropic tendency. The macrophage infection capability of the two viruses did not reflect differences in the ability to either induce or inhibit nitric oxide production. Moderate increases in the ARV-138 multiplicity of infection resulted in a concomitant increase in macrophage infection, and under such conditions the kinetics and extent of the ARV-138 replication cycle were equivalent to those of the highly infectious ARV-176 strain. These results indicated that both viruses are apparently equally capable of replicating in an infected macrophage, but they differ in the ability to establish productive infections in these cells. Using a genetic reassortant approach, we determined that the macrophagotropic property of ARV-176 reflects a post-receptor-binding step in the virus replication cycle and that the ARV-176 M2 genome segment is required for efficient infection of HD-11 cells. The M2 genome segment encodes the major mu-class outer capsid protein (muB) of the virus, which is involved in virus entry and transcriptase activation, suggesting that a host-specific influence on ARV entry and/or uncoating may affect the likelihood of the virus establishing a productive infection in a macrophage cell.

Reovirus sigmaNS protein is required for nucleation of viral assembly complexes and formation of viral inclusions.

Progeny virions of mammalian reoviruses are assembled in the cytoplasm of infected cells at discrete sites termed viral inclusions. Studies of temperature-sensitive (ts) mutant viruses indicate that nonstructural protein sigmaNS and core protein mu2 are required for synthesis of double-stranded (ds) RNA, a process that occurs at sites of viral assembly. We used confocal immunofluorescence microscopy and ts mutant reoviruses to define the roles of sigmaNS and mu2 in viral inclusion formation. In cells infected with wild-type (wt) reovirus, sigmaNS and mu2 colocalize to large, perinuclear structures that correspond to viral inclusions. In cells infected at a nonpermissive temperature with sigmaNS-mutant virus tsE320, sigmaNS is distributed diffusely in the cytoplasm and mu2 is contained in small, punctate foci that do not resemble viral inclusions. In cells infected at a nonpermissive temperature with mu2-mutant virus tsH11.2, mu2 is distributed diffusely in the cytoplasm and the nucleus. However, sigmaNS localizes to discrete structures in the cytoplasm that contain other viral proteins and are morphologically indistinguishable from viral inclusions seen in cells infected with wt reovirus. Examination of cells infected with wt reovirus over a time course demonstrates that sigmaNS precedes mu2 in localization to viral inclusions. These findings suggest that viral RNA-protein complexes containing sigmaNS nucleate sites of viral replication to which other viral proteins, including mu2, are recruited to commence dsRNA synthesis.

A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.

Freon 113 (Freon) is an essential component used in some viral purification methods to separate virus from infected cell debris. With its environmental and toxic hazards, Freon's availability is limited and more tightly regulated. Several organic solvent substitutes were selected to identify a suitable Freon replacement for the purification of both cultivable reovirus and fastidious calicivirus. Reovirus was extracted from tissue cultured cells with each solvent tested and purified in cesium chloride gradients by standard techniques. Purified virions were analyzed for conservation of physical and biological properties by morphological examination and infectivity studies. The purification of calicivirus nucleic acid from stool samples using selected solvents was also examined. Solvent-extracted calicivirus RNA was reverse transcribed and quantified by polymerase chain reaction amplification of a standard diagnostic 117 bp amplicon. These studies indicated that Vertrel XF (a newly developed environmentally friendly Freon substitute) and a 7:3 mixture of isopentane/1-chlorobutane are suitable replacements. Considerations of flammability and ease of use suggest that Vertrel XF is the preferred choice as a Freon substitute for the purification of these non-enveloped viruses.

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers.

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.

The reovirus mutant tsA279 L2 gene is associated with generation of a spikeless core particle: implications for capsid assembly.

Previous studies which used intertypic reassortants of the wild-type reovirus serotype 1 Lang and the temperature-sensitive (ts) serotype 3 mutant clone tsA279 identified two ts lesions; one lesion, in the M2 gene segment, was associated with defective transmembrane transport of restrictively assembled virions (P. R. Hazelton and K. M. Coombs, Virology 207:46-58, 1995). In the present study we show that the second lesion, in the L2 gene segment, which encodes the lambda2 protein, is associated with the accumulation of a core-like particle defective for the lambda2 pentameric spike. Physicochemical, biochemical, and immunological studies showed that these structures were deficient for genomic double-stranded RNA, the core spike protein lambda2, and the minor core protein micro2. Core particles with the lambda2 spike structure accumulated after temperature shift-down from a restrictive to a permissive temperature in the presence of cycloheximide. These data suggest the spike-deficient, core-like particle is an assembly intermediate in reovirus morphogenesis. The existence of this naturally occurring primary core structure suggests that the core proteins lambda1, lambda3, and sigma2 interact to initiate the process of virion capsid assembly through a dodecahedral mechanism. The next step in the proposed capsid assembly model would be the association of the minor core protein mu2, either preceding or collateral to the condensation of the lambda2 pentameric spike at the apices of the primary core structure. The assembly pathway of the reovirus double capsid is further elaborated when these observations are combined with structures identified in other studies.

Absence of superinfection exclusion during asynchronous reovirus infections of mouse, monkey, and human cell lines.

Reovirus is a gastroenteric virus with a genome that consists of ten segments of double-stranded RNA. The segmented nature of the genome allows for genetic mixing when cells are simultaneously infected with two different viral serotypes. The ability of viral reassortment to take place in asynchronous infections has not previously been investigated with mammalian reoviruses. In this study, five different cell lines, representing mouse, monkey, and human, were infected synchronously or asynchronously with various sets of two different temperature-sensitive (ts) reovirus mutants in order to study the genetic interactions which occur. Recombinant viruses were detected at high frequency when infection by the two different ts mutants was separated by as much as 24 h, suggesting that superinfection exclusion does not play a role in reovirus mixed infections. The apparent lack of superinfection exclusion in reovirus infections may have important implications in its evolution.

Stoichiometry of reovirus structural proteins in virus, ISVP, and core particles.

All eight reovirus structural proteins were resolved in a new tris, glycine, and urea (TGU) electrophoretic gel system. The specific identities of proteins were determined immunologically, biochemically, and genetically. Structural proteins of reovirus type 1 Lang had different mobilities in the TGU gel than did type 3 Dearing proteins. Intertypic reassortant viruses that contained various combinations of parental genes were used to identify each of the viral protein bands. Type 1 Lang virions were metabolically-labelled with either 3H-amino acids or 35S-methionine/cysteine and gradient purified. Aliquots of purified virions were treated to generate infectious subviral particles (ISVPs) and core particles. Radiolabelled virus, ISVP, and core proteins were resolved in the TGU gel and protein band intensities were used to determine copy numbers of each structural protein. These studies confirmed the copy numbers and locations of most reovirus proteins. However, important new findings include the discovery that virions contain approximately 120 copies of major core protein sigma 2 and 20 copies of the polymerase cofactor protein mu 2, and ISVP particles contain about 24 copies of mu 1 C that has not been processed to the delta peptide. These data are used to generate a new model of the arrangement of structural proteins with the reovirus particle.

In celebration of the 200th anniversary of Edward Jenner's Inquiry into the causes and effects of the variolae vaccinae.

This review commemorates the 200th anniversary of Edward Jenner's development of a vaccine for variola, the cause of smallpox, and the 20th anniversary of its eradication. Jenner's original 23 case reports are briefly revisited within the context of earlier attempts to prevent this dreaded disease and in light of the current understanding of vaccinology and immunology. In addition, with molecular biological information available about many pox viruses and detailed sequence knowledge of some, it is now possible to appreciate Jenner's prescient accomplishments more fully.

Assembly of the reovirus outer capsid requires mu 1/sigma 3 interactions which are prevented by misfolded sigma 3 protein in temperature-sensitive mutant tsG453.

A temperature-sensitive reovirus mutant, tsG453, whose defect was mapped to major outer capsid protein sigma 3, makes core particles but fails to assemble the outer capsid around the core at non-permissive temperature. Previous studies that made use of electron cryo-microscopy and image reconstructions showed that mu 1, the other major outer capsid protein, but not sigma 3, interact extensively with the core capsid. Although wild-type sigma 3 and mu 1 interact with each other, immunocoprecipitation studies showed that mutant sigma 3 protein was incapable of interacting with mu 1 at the non-permissive temperature. In addition, restrictively-grown mutant sigma 3 protein could not be precipitated by some sigma 3-specific monoclonal antibodies. These observations suggest that in a wild-type infection, specific sigma 3 and mu 1 interactions result in changes in mu 1 conformation which are required to allow mu 1/sigma 3 complexes to condense onto the core capsid shell during outer capsid assembly, and that sigma 3 in non-permissive tsG453 infections is misfolded such that it cannot interact with mu 1.

Nonrandom segregation of parental alleles in reovirus reassortants.

To test for nonrandom segregations among their 10 genomic RNA segments, we examined a set of 83 reassortants derived from mammalian reovirus type 1 Lang and type 3 Dearing. After confirming the genotypes of the reassortants, we performed statistical analyses on the distributions of parental alleles for each of the 10 gene segments, as well as for the 45 possible pairings of the 10 segments. The analyses revealed nonrandom associations of parental alleles in the L1-L2, L1-M1, L1-S1, and L3-S1 segment pairs, at levels indicating high statistical significance (P < 0.005). Such associations may reflect specific interactions between viral components (protein-protein, protein-RNA, or RNA-RNA) and may influence both the evolution of reoviruses in nature and their genetic analysis in the laboratory. The data may also support an hypothesis that reovirus reassortants commonly contain mutations that improve their fitness for independent replication.

Identification and characterization of a double-stranded RNA- reovirus temperature-sensitive mutant defective in minor core protein mu2.

A newly identified temperature-sensitive mutant whose defect was mapped to the reovirus M1 gene (minor core protein mu2) was studied to better understand the functions of this virion protein. Sequence determination of the Ml gene of this mutant (tsH11.2) revealed a predicted methionine-to-threonine alteration at amino acid 399 and a change from proline to histidine at amino acid 414. The mutant made normal amounts of single-stranded RNA, both in in vitro transcriptase assays and in infected cells, and normal amounts of progeny viral protein at early times in a restrictive infection. However, tsH11.2 produced neither detectable progeny protein nor double-stranded RNA at late times in a restrictive infection. These studies indicate that mu2 plays a role in the conversion of reovirus mRNA to progeny double-stranded RNA.

The M1 gene is associated with differences in the temperature optimum of the transcriptase activity in reovirus core particles.

The reovirus core is a multienzyme complex that contains five different structural proteins and 10 segments of double-stranded RNA. The core is responsible for transcribing mRNA from the enclosed double-stranded RNA. The reovirus transcriptase has an unusual temperature profile, with optimum transcription occurring at approximately 50 degrees C and little activity occurring below 30 or above 60 degrees C. Purified reovirus serotype 1 Lang (T1L) cores transcribed most efficiently at 48 degrees C. The transcriptase temperature optimum of purified reovirus serotype 3 Dearing (T3D) cores was 52 degrees C. In addition, T1L cores produced more mRNA per particle than did T3D cores at their respective temperature optima. Core particles were purified from T1L x T3D reassortants and were used to map these differences. The M1 gene, which encodes minor core protein mu 2, was uniquely associated with the difference in temperature optimum of transcription (P = 0.0003). The L1 gene, which encodes minor core protein lambda 3 (previously implicated as the RNA polymerase), and the M1 gene were associated with the difference in absolute amounts of transcript produced (P = 0.01 and P = 0.0002, respectively). These data suggest that minor core protein mu 2 also plays a role in reovirus transcription.

The reovirus mutant tsA279 has temperature-sensitive lesions in the M2 and L2 genes: the M2 gene is associated with decreased viral protein production and blockade in transmembrane transport.

Temperature-sensitive mutants provide an ideal means for dissecting viral assembly pathways. The morphological variants produced by and biological characteristics of tsA279, a previously uncharacterized mutant from the Fields' panel of temperature-sensitive mutants of reovirus, were determined under restrictive growth conditions. The mutant showed a distinctive pattern of increased temperature sensitivity as the temperature was raised from 39 degrees to 40 degrees. Wild-type reovirus type 1 Lang and the mutant were crossed to generate reassortants. Efficiency of plating analyses of the reassortants showed that tsA279 has temperature-sensitive lesions in two genes, a mildly temperature-sensitive one in L2, which encodes core spike protein lambda 2, and a stronger, dominant lesion in M2, which encodes major outer capsid protein mu 1. Electron microscopic examination of thin-sectioned tsA279-infected cells showed three ways in which the mutant phenotypes were expressed. The mutant appeared to be blocked in transmembrane transport of virions, a phenotype that mapped to the M2 gene; the mutant produced significantly reduced amounts of identifiable particles; and those particles that were produced appeared to be morphological variants. Immunofluorescent microscopy and immunoprecipitations of tsA279- and various T1L x tsA279 reassortant-infected cells suggested that the reduction in observed progeny was caused by a decreased production of viral proteins at the nonpermissive temperature. This phenotype also mapped to the mutant M2 gene.

Studies of the major reovirus core protein sigma 2: reversion of the assembly-defective mutant tsC447 is an intragenic process and involves back mutation of Asp-383 to Asn.

The reovirus group C temperature-sensitive mutant tsC447, whose defect maps to the S2 gene, which encodes the major core protein sigma 2, fails to assemble core particles at the nonpermissive temperature. To identify other proteins that may interact with sigma 2 during assembly, we generated and examined 10 independent revertants of the mutant. To determine which gene(s) carried a compensatory suppressor mutation(s), we generated intertypic reassortants between wild-type reovirus serotype 1 Lang and each revertant and determined the temperature sensitivities of the reassortants by efficiency-of-plating assays. Results of the efficiency-of-plating analyses indicated that reversion of the tsC447 defect was an intragenic process in all revertants. To identify the region(s) of sigma 2 that had reverted, we determined the nucleotide sequences of the S2 genes. In all revertant sequences examined, the G at nucleotide position 1166 in tsC447 had reverted to the A present in the wild-type sequence. This reversion leads to the restoration of a wild-type asparagine (in place of a mutant aspartic acid) at amino acid 383 in the sigma 2 sequence. These results collectively indicate that the functional lesion in tsC447 is Asp-383 and that this lesion cannot be corrected by alterations in other core proteins. These observations suggest that this region of sigma 2, which may be important in mediating assembly of the core particle, does not interact significantly with other reovirus proteins.