Leslie A. Schiff

Reovirus Induces and Benefits from an Integrated Cellular Stress Response

ABSTRACT Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58IPK, the cellular inhibitor of the eukaryotic initiation factor 2α (eIF2α) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2α phosphorylation and unphosphorylated eIF2α is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2α phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2α kinases and phosphorylatable eIF2α. When eIF2α is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2α phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2α phosphorylation facilitates reovirus replication in two ways—first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.

Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.

By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.

Proteolytic disassembly is a critical determinant for reovirus oncolysis.

See page 1406 Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

The cellular protein P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism.

ABSTRACT We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58IPK, the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2α. P58IPK also inhibits PERK, an eIF2α kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58IPK to interact with and inhibit multiple eIF2α kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58IPK during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58IPK−/− mice, we demonstrated that the absence of P58IPK led to an increase in eIF2α phosphorylation and decreased influenza virus mRNA translation. The absence of P58IPK also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58IPK target, PKR, the trends were reversed—eIF2α phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58IPK also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2α phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2α. Taken together, our results support a model in which P58IPK regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

Structure of the reovirus outer capsid and dsRNA-binding protein σ3 at 1.8 Å resolution

The crystallographically determined structure of the reovirus outer capsid protein σ3 reveals a two‐lobed structure organized around a long central helix. The smaller of the two lobes includes a CCHC zinc‐binding site. Residues that vary between strains and serotypes lie mainly on one surface of the protein; residues on the opposite surface are conserved. From a fit of this model to a reconstruction of the whole virion from electron cryomicroscopy, we propose that each σ3 subunit is positioned with the small lobe anchoring it to the protein μ1 on the surface of the virion, and the large lobe, the site of initial cleavages during entry‐related proteolytic disassembly, protruding outwards. The surface containing variable residues faces solvent. The crystallographic asymmetric unit contains two σ3 subunits, tightly associated as a dimer. One broad surface of the dimer has a positively charged surface patch, which extends across the dyad. In infected cells, σ3 binds dsRNA and inhibits the interferon response. The location and extent of the positively charged surface patch suggest that the dimer is the RNA‐binding form of σ3.

Addition of Exogenous Protease Facilitates Reovirus Infection in Many Restrictive Cells

ABSTRACT Virion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.

Cathepsin S supports acid-independent infection by some reoviruses

In murine fibroblasts, efficient proteolysis of reovirus outer capsid protein σ3 during cell entry by virions requires the acid-dependent lysosomal cysteine protease cathepsin L. The importance of cathepsin L for infection of other cell types is unknown. Here we report that the acid-independent lysosomal cysteine protease cathepsin S mediates outer capsid processing in macrophage-like P388D cells. P388D cells supported infection by virions of strain Lang, but not strain c43. Genetic studies revealed that this difference is determined by S4, the viral gene segment that encodes σ3. c43-derived subvirion particles that lack σ3 replicated normally in P388D cells, suggesting that the difference in infectivity of Lang and c43 virions is at the level of σ3 processing. Infection of P388D cells with Lang virions was inhibited by the broad spectrum cysteine protease inhibitor trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane but not by NH4Cl, which raises the endocytic pH and thereby inhibits acid-dependent proteases such as cathepsins L and B. Outer capsid processing and infection of P388D cells with Lang virions were also inhibited by a cathepsin S-specific inhibitor. Furthermore, in the presence of NH4Cl, cell lines engineered to express cathepsin S supported infection by Lang, but not c43, virions. Our results thus indicate that differences in susceptibility to cathepsin S-mediated σ3 processing are responsible for strain differences in reovirus infection of macrophage-like P388D cells and other cathepsin S-expressing cells. Additionally, our data suggest that the acid dependence of reovirus infections of most other cell types may reflect the low pH requirement for the activities of most other lysosomal proteases rather, than some other acid-dependent aspect of cell entry.

New ways of initiating translation in eukaryotes? [2](multiple letters)

This letter to the editor is a response by a large number of investigators in the field of protein synthesis to the minireview published by Dr. Kozak in Molecular and Cellular Biology (9). This minireview attempts to create significant doubts regarding the published literature that we believe are unwarranted and to bolster Dr. Kozak’s own point of view regarding translation initiation. We therefore take serious issue with the scholarliness of the Kozak minireview. As will be shown, the Kozak minireview contains numerous distortions of fact and of published data and selectively utilizes the published literature. In every field of research there are legitimate concerns regarding the interpretation of results and the reproducibility of certain published data. Several of the issues raised by Dr. Kozak are legitimate in this regard, but they are not new and have hardly gone unnoticed, having been raised in scholarly and critical reviews elsewhere. At issue here is not the right to critically question results and interpretations but rather whether the Kozak minireview is scholarly and its tone is professional. We point out that much of the work challenged in the Kozak minireview was published in Molecular and Cellular Biology, as well as other leading peer-reviewed journals, and forms a mainstream of research on protein synthesis which is taking place in scores of laboratories around the world. In this minireview, Dr. Kozak dismisses three novel mechanisms for translation initiation which have now been well studied and extensively documented. One mechanism is internal ribosome entry, which she rejects in favor of ribosome scanning, a mechanism for translation initiation which she proposed over 20 years ago. In ribosome scanning, it is proposed that the 40S small ribosome subunit enters the mRNA from the 5 cap and undergoes a linear 5 -to-3 search for the initiation codon, which is typically an AUG. Internal ribosome entry involves the internal association of ribosome subunits at or near the initiation codon without the need for entry from the 5 end of the mRNA. A second mechanism opposed by Dr. Kozak is the initiation of protein synthesis without Met-tRNA, a universal and key component, as shown for several insect virus mRNAs. The ability to carry out translation without this initiator tRNA, and from the A site of the ribosome, has enormous implications for our understanding of protein synthesis and its evolution. A third mechanism of translation initiation which Dr. Kozak takes issue with is known as ribosome shunting or discontinuous scanning, which combines features of 5 entry of ribosomes by scanning and the internal translocation of ribosome subunits without further scanning to the initiation codon. It is clear to a great many researchers, as represented by the signatory list below, that the initiation of protein synthesis in eukaryotes is dynamic and flexible, involving a variety of mechanisms that have evolved to meet the complex demands of eukaryotic cells and viruses. Dr. Kozak has spent more than 10 years in strenuous opposition to the evidence for viral internal ribosome entry and the recognition of specific viral cis-acting internal ribosome entry site (IRES) elements. The minireview now attempts to use almost entirely the same kinds of arguments against cellular IRESs and other means of nonscanning translation initiation that Dr. Kozak used previously in her unsuccessful efforts to disprove viral IRESs. Whether Dr. Kozak explicitly acknowledges internal ribosome entry as an established fact, at least for viruses, is not at all clear in the minireview, although she does compare translation functions to the encephalomyocarditis virus (EMCV) IRES, but without comment or acceptance. It would be fairer to the reader and more intellectually honest to explicitly acknowledge internal ribosome entry as an established fact, at least for viruses, or—if she still wishes to oppose the idea—to do so openly. Needless to say, it is now difficult to mount a convincing case for blanket repudiation of IRESs in the face of overwhelming data, including elegant and compelling evidence from viral IRES-dependent translation of a circular RNA (3), which was not cited in the Kozak review. It is not practical to document here all of the examples in which published results were inappropriately presented in the minireview by Dr. Kozak. We refer readers to a recent comprehensive review which summarizes the current evidence in support of viral and cellular IRES elements and alternate mechanisms of translation initiation in eukaryotes and briefly overviews some of the key techniques which were questioned by Dr. Kozak (7). Consequently, we list below just several specific examples which are emblematic of the serious issues which are of concern to us. Several reasons are described by Dr. Kozak for dismissing reports of cellular IRESs. Dr. Kozak argues that because cellular IRESs often represent modest translation increases over background levels, they result from fortuitous positioning of RNA sequences in experimental constructs. This argument ignores the evidence that IRESs have been shown to represent a range of activities from weak to strong and to function by a variety of mechanisms. Indeed, the expression of many cellular genes encoding regulatory proteins is often tightly controlled at multiple steps to guarantee that correct protein levels are achieved, which is not generally equivalent to high protein levels. An IRES may therefore be relatively weak, but in combination with other levels of gene control, it achieves significant or correct protein expression levels under different physiological conditions. One example is the IRES of the proto-oncogene c-sis, which encodes platelet-derived growth factor 2 (PDGF2). This IRES is activated severalfold during megakaryocyte differentiation, in conjunction with induction of PDGF2/c-sis gene expression during differentiation (1, 16). The modest translation stimulation directed by the cellular PDGF2 IRES fine tunes PDGF2/c-sis gene expression during differentiation. Similar mechanisms are likely employed by other critical regulatory genes and may have widespread implications for cellular growth and development. Thus, it is arbitrary to dismiss cellular IRES elements as physiologically irrelevant artifacts merely because their effects on translation are moderate. A more considered view is that regulatory elements act at all levels of gene control, including transcription, mRNA transport, and mRNA stability and translation, and permit exquisite control precisely because they involve multiple and modest additive effects which can be independently combined and regulated. There are several functional ways to study IRESs. Construction of a dicistronic mRNA containing an internal downstream second open reading frame that is ordinarily not translated is typically used to detect IRES activity. Other approaches include insertion of very stable, translation-blocking hairpin structures upstream of the IRES and biochemical detection of IRES interaction with initiation factors and ribosome subunits. Important control studies must be performed to validate the integrity of the dicistronic mRNA and to exclude the presence of cryptic promoters or aberrant splicing that could lead to

Reovirus Uses Multiple Endocytic Pathways for Cell Entry

ABSTRACT Entry of reovirus virions has been well studied in several tissue culture systems. After attachment to junctional adhesion molecule A (JAM-A), virions undergo clathrin-mediated endocytosis followed by proteolytic disassembly of the capsid and penetration to the cytoplasm. However, during in vivo infection of the intestinal tract, and likely in the tumor microenvironment, capsid proteolysis (uncoating) is initiated extracellularly. We used multiple approaches to determine if uncoated reovirus particles, called intermediate subviral particles (ISVPs), enter cells by directly penetrating the limiting membrane or if they take advantage of endocytic pathways to establish productive infection. We found that entry and infection by reovirus ISVPs was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis, as well as by genistein and dominant-negative caveolin-1, which block caveolar endocytosis. Inhibition of caveolar endocytosis also reduced infection by reovirus virions. Extraction of membrane cholesterol with methyl-β-cyclodextrin inhibited infection by virions but had no effect when infection was initiated with ISVPs. We found this pathway to be independent of both clathrin and caveolin. Together, these data suggest that reovirus virions can use both dynamin-dependent and dynamin-independent endocytic pathways during cell entry, and they reveal that reovirus ISVPs can take advantage of caveolar endocytosis to establish productive infection.

Involvement of the Interferon-Regulated Antiviral Proteins PKR and RNase L in Reovirus-Induced Shutoff of Cellular Translation

ABSTRACT Cellular translation is inhibited following infection with most strains of reovirus, but the mechanisms responsible for this phenomenon remain to be elucidated. The extent of host shutoff varies in a strain-dependent manner; infection with the majority of strains leads to strong host shutoff, while infection with strain Dearing results in minimal inhibition of cellular translation. A genetic study with reassortant viruses and subsequent biochemical analyses led to the hypothesis that the interferon-induced, double-stranded RNA-activated protein kinase, PKR, is responsible for reovirus-induced host shutoff. To directly determine whether PKR is responsible for reovirus-induced host shutoff, we used a panel of reovirus strains and mouse embryo fibroblasts derived from knockout mice. This approach revealed that PKR contributes to but is not wholly responsible for reovirus-induced host shutoff. Studies with cells lacking RNase L, the endoribonuclease component of the interferon-regulated 2′,5′-oligoadenylate synthetase-RNase L system, demonstrated that RNase L also down-regulates cellular protein synthesis in reovirus-infected cells. In many viral systems, PKR and RNase L have well-characterized antiviral functions. An analysis of reovirus replication in cells lacking these molecules indicated that, while they contributed to host shutoff, neither PKR nor RNase L exerted an antiviral effect on reovirus growth. In fact, some strains of reovirus replicated more efficiently in the presence of PKR and RNase L than in their absence. Data presented in this report illustrate that the inhibition of cellular translation following reovirus infection is complex and involves multiple interferon-regulated gene products. In addition, our results suggest that reovirus has evolved effective mechanisms to avoid the actions of the interferon-stimulated antiviral pathways that include PKR and RNase L and may even benefit from their expression.