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Many colleges and universities utilized wastewater surveillance testing for SARS-CoV-2 RNA as a tool to help monitor and mitigate the COVID-19 pandemic on campuses across the USA during the 2020–2021 academic year. We sought to assess the efficacy of one such program by analyzing data on relative wastewater RNA levels from residential buildings in relation to SARS-CoV-2 cases identified through individual surveillance testing, conducted largely independent of wastewater results. Almost 80% of the cases on campus were associated with positive wastewater tests, resulting in an overall positive predictive value of 79% (Chi square 48.1, Df = 1, P

Following the initial detection of viral infection, innate immune responses trigger the induction of numerous interferon-stimulated genes (ISGs) to inhibit virus replication and dissemination. One such ISG encodes cholesterol-25-hydroxylase (CH25H), an enzyme that catalyzes the oxidation of cholesterol to form a soluble product, 25-hydroxycholesterol (25HC). Recent studies have found that CH25H is broadly antiviral; it inhibits infection by several viruses. For enveloped viruses, 25HC inhibits membrane fusion, likely by altering membrane characteristics such as hydrophobicity or cholesterol aggregation. However, the mechanisms by which 25HC restricts infection of nonenveloped viruses are unknown. We examined whether 25HC restricts infection by mammalian reovirus. Treatment with 25HC restricted infection by reovirus prototype strains type 1 Lang and type 3 Dearing. In contrast to reovirus virions, 25HC did not restrict infection by reovirus infectious subvirion particles (ISVPs), which can penetrate either directly at the cell surface or in early endosomal membranes. Treatment with 25HC altered trafficking of reovirus particles to late endosomes and delayed the kinetics of reovirus uncoating. These results suggest that 25HC inhibits the efficiency of cellular entry of reovirus virions, which may require specific endosomal membrane dynamics for efficient membrane penetration. IMPORTANCE The innate immune system is crucial for effective responses to viral infection. Type I interferons, central components of innate immunity, induce expression of hundreds of ISGs; however, the mechanisms of action of these antiviral proteins are not well understood. CH25H, encoded by an ISG, represents a significant constituent of these cellular antiviral strategies, as its metabolic product, 25HC, can act in both an autocrine and a paracrine fashion to protect cells from infection and has been shown to limit viral infection in animal models. Further investigation into the mechanism of action of 25HC may inform novel antiviral therapies and influence the use of mammalian reovirus in clinical trials as an oncolytic agent.

Serotype 3 (T3) reoviruses induce substantially more type 1 interferon (IFN-I) secretion than serotype 1 (T1) strains. However, the mechanisms underlying differences in IFN-I production between T1 and T3 reoviruses remain undefined. Here, we found that differences in IFN-I production between T1 and T3 reoviruses correlate with activation of interferon regulatory factor 3 (IRF3), a key transcription factor for the production of IFN-I. T3 strain rsT3D activated IRF3 more rapidly and to a greater extent than the T1 strain rsT1L, in simian virus 40 (SV40) immortalized endothelial cells (SVECs). Differences in IRF3 activation between rsT1L and rsT3D were observed in the first hours of infection and were independent of de novo viral RNA and protein synthesis. NF-κB activation mirrored IRF3 activation, with rsT3D inducing more NF-κB activity than rsT1L. We also found that IRF3 and NF-κB are activated in a mitochondrial antiviral-signaling protein (MAVS)-dependent manner. rsT1L does not suppress IRF3 activation, as IRF3 phosphorylation could be induced in rsT1L-infected cells. Transfected rsT1L and rsT3D RNA induced IRF3 phosphorylation, indicating that genomic RNA from both strains has the capacity to activate IRF3. Finally, bypassing the normal route of reovirus entry by transfecting in vitro -generated viral cores revealed that rsT1L and rsT3D core particles induced equivalent IRF3 activation. Taken together, our findings indicate that entry-related events that occur after outer capsid disassembly, but prior to deposition of viral cores into the cytoplasm, influence the efficiency of IFN-I responses to reovirus. This work provides further insight into mechanisms by which nonenveloped viruses activate innate immune responses. IMPORTANCE Detection of viral nucleic acids by the host cell triggers type 1 interferon (IFN-I) responses, which are critical for containing and clearing viral infections. Viral RNA is sensed in the cytoplasm by cellular receptors that initiate signaling pathways, leading to the activation of interferon regulatory factor 3 (IRF3) and NF-κB, key transcription factors required for IFN-I induction. Serotype 3 (T3) reoviruses induce significantly more IFN-I than serotype 1 (T1) strains. In this work, we found that differences in IFN-I production by T1 and T3 reoviruses correlate with differential IRF3 activation. Differences in IRF3 activation are not caused by a blockade of the IRF3 activation by a T1 strain. Rather, differences in events during the late stages of viral entry determine the capacity of reovirus to activate host IFN-I responses. Together, our work provides insight into mechanisms of IFN-I induction by nonenveloped viruses.

Reoviruses are double-stranded RNA viruses that infect the mammalian respiratory and gastrointestinal tract. Reovirus infection elicits production of type I interferons (IFNs), which trigger antiviral pathways through the induction of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified, the functions of many of these genes are unknown. The interferon-inducible transmembrane (IFITM) proteins are one class of ISGs that restrict the cell entry of some enveloped viruses, including influenza A virus. One family member, IFITM3, localizes to late endosomes, where reoviruses undergo proteolytic disassembly; therefore, we sought to determine whether IFITM3 also restricts reovirus entry. IFITM3-expressing cell lines were less susceptible to infection by reovirus, as they exhibited significantly lower percentages of infected cells in comparison to control cells. Reovirus replication was also significantly reduced in IFITM3-expressing cells. Additionally, cells expressing an shRNA targeting IFITM3 exhibited a smaller decrease in infection after IFN treatment than the control cells, indicating that endogenous IFITM3 restricts reovirus infection. However, IFITM3 did not restrict entry of reovirus infectious subvirion particles (ISVPs), which do not require endosomal proteolysis, indicating that restriction occurs in the endocytic pathway. Proteolysis of outer capsid protein μ1 was delayed in IFITM3-expressing cells in comparison to control cells, suggesting that IFITM3 modulates the function of late endosomal compartments either by reducing the activity of endosomal proteases or delaying the proteolytic processing of virions. These data provide the first evidence that IFITM3 restricts infection by a nonenveloped virus and suggest that IFITM3 targets an increasing number of viruses through a shared requirement for endosomes during cell entry.

A variety of signal transduction pathways are activated in response to viral infection, which dampen viral replication and transmission. These mechanisms involve both the induction of type I interferons (IFNs), which evoke an antiviral state, and the triggering of apoptosis. Mammalian orthoreoviruses are double-stranded RNA viruses that elicit apoptosis in vitro and in vivo . The transcription factors interferon regulatory factor 3 (IRF-3) and nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) are required for the expression of IFN-β and the efficient induction of apoptosis in reovirus-infected cells. However, it is not known whether IFN-β induction is required for apoptosis, nor have the genes induced by IRF-3 and NF-κB that are responsible for apoptosis been identified. To determine whether IFN-β is required for reovirus-induced apoptosis, we used type I IFN receptor-deficient cells, IFN-specific antibodies, and recombinant IFN-β. We found that IFN synthesis and signaling are dispensable for the apoptosis of reovirus-infected cells. These results indicate that the apoptotic response following reovirus infection is mediated directly by genes responsive to IRF-3 and NF-κB. Noxa is a proapoptotic BH3-domain-only protein of the Bcl-2 family that requires IRF-3 and NF-κB for efficient expression. We found that Noxa is strongly induced at late times (36 to 48 h) following reovirus infection in a manner dependent on IRF-3 and NF-κB. The level of apoptosis induced by reovirus is significantly diminished in cells lacking Noxa, indicating a key prodeath function for this molecule during reovirus infection. These results suggest that prolonged innate immune response signaling induces apoptosis by eliciting Noxa expression in reovirus-infected cells.

The secreted cytokine alpha/beta interferon (IFN-α/β) binds its receptor to activate the Jak-STAT signal transduction pathway, leading to formation of the heterotrimeric IFN-stimulated gene factor 3 (ISGF3) transcription complex for induction of IFN-stimulated genes (ISGs) and establishment of an antiviral state. Many viruses have evolved countermeasures to inhibit the IFN pathway, thereby subverting the innate antiviral response. Here, we demonstrate that the mildly myocarditic reovirus type 1 Lang (T1L), but not the nonmyocarditic reovirus type 3 Dearing, represses IFN induction of a subset of ISGs and that this repressor function segregates with the T1L M1 gene. Concordantly, the T1L M1 gene product, μ2, dramatically inhibits IFN-β-induced reporter gene expression. Surprisingly, T1L infection does not degrade components of the ISGF3 complex or interfere with STAT1 or STAT2 nuclear translocation as has been observed for other viruses. Instead, infection with T1L or reassortant or recombinant viruses containing the T1L M1 gene results in accumulation of interferon regulatory factor 9 (IRF9) in the nucleus. This effect has not been previously described for any virus and suggests that μ2 modulates IRF9 interactions with STATs for both ISGF3 function and nuclear export. The M1 gene is a determinant of virus strain-specific differences in the IFN response, which are linked to virus strain-specific differences in induction of murine myocarditis. We find that virus-induced myocarditis is associated with repression of IFN function, providing new insights into the pathophysiology of this disease. Together, these data provide the first report of an increase in IRF9 nuclear accumulation associated with viral subversion of the IFN response and couple virus strain-specific differences in IFN antagonism to the pathogenesis of viral myocarditis.

Heterozygosity for the CCR5 Delta32 allele is associated with delayed progression to AIDS in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an unusual HIV-1 isolate from the blood of an asymptomatic individual who was heterozygous for the CCR5 Delta32 allele and had reduced levels of CCR5 expression. The primary virus used CCR5, CXCR4, and an unusually broad range of alternative coreceptors to enter transfected cells. However, only CXCR4 and CCR5 were used to enter primary T cells and monocyte-derived macrophages, respectively. Full-length Env clones had an unusually long V1/V2 region and rare amino acid variants in the V3 and C4 regions. Mutagenesis studies and structural models suggested that Y308, D321, and to a lesser extent K442 and E444, contribute to the broad coreceptor usage of these Envs, whereas I317 is likely to be a compensatory change. Furthermore, database analysis suggests that covariation can occur at positions 308/317 and 308/321 in vivo. Y308 and D321 reduced dependence on the extracellular loop 2 (ECL2) region of CCR5, while these residues along with Y330, K442, and E444 enhanced dependence on the CCR5 N-terminus compared to clade B consensus residues at these positions. These results suggest that expanded coreceptor usage of HIV-1 can occur in some individuals without rapid progression to AIDS as a consequence of changes in the V3 region that reduce dependence on the ECL2 region of CCR5 by enhancing interactions with conserved structural elements in G-protein-coupled receptors.

Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. Point mutations introduced into viral capsid proteins σ1 and σ3 were used to define sequences that govern susceptibility to cleavage by intestinal proteases. We recovered a recombinant virus that expresses green fluorescent protein (GFP) by replacement of the σ3 open-reading frame with GFP. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4+and CD8+T cells. Infection of primary T-cell cultures with ELI6 induced CD4+T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4+and CD8+T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4+T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8+T cells was triggered by a soluble factor(s) secreted by CD4+T cells. HIV-1 virions activated CD4+and CD8+T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25+HLA-DR+T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4+and CD8+T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.

Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.

Mammalian orthoreoviruses (reoviruses) are members of the Reoviridae. Reoviruses contain 10 double-stranded (ds) RNA gene segments enclosed in two concentric protein shells, called outer capsid and core. These viruses serve as a versatile experimental system for studies of viral replication events at the virus-cell interface, including engagement of cell-surface receptors, internalization and disassembly, and activation of the innate immune response, including NF-κB-dependent cellular signaling pathways. Reoviruses also provide a model system for studies of virus-induced apoptosis and organ-specific disease in vivo.Reoviruses attach to host cells via the filamentous attachment protein, σ1. The σ1 protein of all reovirus serotypes engages junctional adhesion molecule-A (JAM-A), an integral component of intercellular tight junctions. The σ1 protein also binds to cell-surface carbohydrate, with the type of carbohydrate bound varying by serotype. Following attachment to JAM-A and carbohydrate, reovirus internalization is mediated by β1 integrins, most likely via clathrin-dependent endocytosis. In the endocytic compartment, reovirus outer-capsid protein σ3 is removed by acid-dependent cysteine proteases in most cell types. Removal of σ3 results in the exposure of a hydrophobic conformer of the viral membrane-penetration protein, μ1, which pierces the endosomal membrane and delivers transcriptionally active reovirus core particles into the cytoplasm.Reoviruses induce apoptosis in both cultured cells and infected mice. Perturbation of reovirus disassembly using inhibitors of endosomal acidification or protease activity abrogates apoptosis. The μ1-encoding M2 gene is genetically linked to strain-specific differences in apoptosis-inducing capacity, suggesting a function for μ1 in induction of death signaling. Reovirus disassembly leads to activation of transcription factor NF-κB, which modulates apoptotic signaling in numerous types of cells. Inhibition of NF-κB nuclear translocation using either pharmacologic agents or expression of transdominant forms of IκB blocks reovirus-induced apoptosis, suggesting an essential role for NF-κB activation in the death response. Multiple effector pathway s downstream of NF-κB-directed gene expression execute reovirus-induced cell death. This chapter will focus on the mechanisms by which reovirus attachment and disassembly activate NF-κB and stimulate the cellular proapoptotic machinery.

Reovirus infection leads to apoptosis in both cultured cells and the murine central nervous system (CNS). NF-κB-driven transcription of proapoptotic cellular genes is required for the effector phase of the apoptotic response. Although both extrinsic death-receptor signaling pathways and intrinsic pathways involving mitochondrial injury are implicated in reovirus-induced apoptosis, mechanisms by which either of these pathways are activated and their relationship to NF-κB signaling following reovirus infection are unknown. The proapoptotic Bcl-2 family member, Bid, is activated by proteolytic cleavage following reovirus infection. To understand how reovirus integrates host signaling circuits to induce apoptosis, we examined proapoptotic signaling following infection of Bid-deficient cells. Although reovirus growth was not affected by the absence of Bid, cells lacking Bid failed to undergo apoptosis. Furthermore, we found that NF-κB activation is required for Bid cleavage and subsequent proapoptotic signaling. To examine the functional significance of Bid-dependent apoptosis in reovirus disease, we monitored fatal encephalitis caused by reovirus in the presence and absence of Bid. Survival of Bid-deficient mice was significantly enhanced in comparison to wild-type mice following either peroral or intracranial inoculation of reovirus. Decreased reovirus virulence in Bid-null mice was accompanied by a reduction in viral yield. These findings define a role for NF-κB-dependent cleavage of Bid in the cell death program initiated by viral infection and link Bid to viral virulence., Author Summary Viruses injure host tissues by activating signaling pathways that trigger cell death by a process called apoptosis. Hence, blockade of apoptosis may serve as a useful strategy to dampen the severity of viral disease. However, deployment of such a strategy requires identification of host signaling networks that control cell death and a detailed molecular blueprint of how these pathways are activated by a virus. In this study, we used mammalian reovirus, an important experimental model for studies of viral encephalitis, to elucidate how cell death pathways are activated following viral infection and whether these signaling cascades influence the capacity of a virus to produce lethal CNS disease. We found that Bid, a host regulator of cell death, influences apoptosis induction by reovirus. Moreover, Bid is required for efficient reovirus replication in the CNS and modulates reovirus neurological disease. These findings highlight Bid as a critical regulator of viral pathogenesis and illuminate a potential new target for development of antiviral therapeutics.

Apoptosis is a pathological hallmark of encephalitis and myocarditis caused by reovirus in newborn mice. In cell culture models, the antiviral transcription factor interferon regulatory factor 3 (IRF-3) enhances reovirus-induced apoptosis following activation via retinoic acid inducible gene I and interferon promoter-stimulating factor 1. To determine the role of IRF-3 in reovirus disease, we infected newborn IRF-3+/+and IRF-3−/−mice perorally with mildly virulent strain type 1 Lang (T1L) and fully virulent strain type 3 SA+ (T3SA+) and monitored infected animals for survival. Both wild-type and IRF-3−/−mice succumbed with equivalent frequencies to infection with T3SA+. However, the absence of IRF-3 was associated with significantly decreased survival rates following infection with T1L. The two virus strains achieved similar peak titers in IRF-3+/+and IRF-3−/−mice in the intestine, brain, heart, liver, and spleen. However, by day 12 postinoculation, titers in all organs examined were 10- to 100-fold higher in IRF-3−/−mice than those in wild-type mice. Increased titers were associated with marked pathological changes in all organs examined, especially in the heart, where absence of IRF-3 resulted in severe myocarditis. Cellular and humoral immune responses were equivalent in wild-type and IRF-3−/−animals, suggesting that IRF-3 functions independently of the adaptive immune response to enhance reovirus clearance. Thus, IRF-3 serves to facilitate virus clearance and prevent tissue injury in response to reovirus infection.

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis and myocarditis in infected animals. Differences in apoptosis efficiency displayed by reovirus strains are linked to the viral μ1-encoding M2 gene segment. Studies using pharmacologic inhibitors of reovirus replication demonstrate that apoptosis induction by reovirus requires viral disassembly in cellular endosomes but not RNA synthesis. Since the μ1 protein functions to pierce endosomal membranes during this temporal window, these findings point to an important role for μ1 in activating signaling pathways that lead to apoptosis. To understand mechanisms used by μ1 to induce apoptosis, a panel of μ1 mutant viruses generated by reverse genetics was analyzed for the capacities to penetrate host cell membranes, activate proapoptotic signaling pathways, evoke cell death, and produce encephalitis in newborn mice. We found that single amino acid changes within the δ region of μ1 reduce the efficiency of membrane penetration. These mutations also diminish the capacities of reovirus to activate proapoptotic transcription factors NF-κB and IRF-3 and elicit apoptosis. Additionally, we observed that following intracranial inoculation, an apoptosis-deficient μ1 mutant is less virulent in newborn mice in comparison to the wild-type virus. These results indicate a critical function for the membrane penetration activity of μ1 in evoking prodeath signaling pathways that regulate reovirus pathogenesis.

During viral infection, cells initiate antiviral responses to contain replication and inhibit virus spread. One protective mechanism involves activation of transcription factors interferon regulatory factor-3 (IRF-3) and NF-kappaB, resulting in secretion of the antiviral cytokine, interferon-beta. Another is induction of apoptosis, killing the host cell before virus disseminates. Mammalian reovirus induces both interferon-beta and apoptosis, raising the possibility that both pathways are initiated by a common cellular sensor. We show here that reovirus activates IRF-3 with kinetics that parallel the activation of NF-kappaB, a known mediator of reovirus-induced apoptosis. Activation of IRF-3 requires functional retinoic acid inducible gene-I and interferon-beta promoter stimulator-1, but these intracellular sensors are dispensable for activation of NF-kappaB. Interferon-beta promoter stimulator-1 and IRF-3 are required for efficient apoptosis following reovirus infection, suggesting a common mechanism of antiviral cytokine induction and activation of the cell death response.

Junctional adhesion molecule-A (JAM-A) serves as a serotype-independent receptor for mammalian orthoreoviruses (reoviruses). The membrane-distal immunoglobulin-like D1 domain of JAM-A is required for homodimerization and binding to reovirus attachment protein sigma1. We employed a structure-guided mutational analysis of the JAM-A dimer interface to identify determinants of reovirus binding. We purified mutant JAM-A ectodomains for solution-phase and surface plasmon resonance binding studies and expressed mutant forms of full-length JAM-A in Chinese hamster ovary cells to assess reovirus binding and infectivity. Mutation of residues in the JAM-A dimer interface that participate in salt-bridge or hydrogen-bond interactions with apposing JAM-A monomers abolishes the capacity of JAM-A to form dimers. JAM-A mutants incapable of dimer formation form complexes with the sigma1 head that are indistinguishable from wild-type JAM-A-sigma1 head complexes, indicating that sigma1 binds to JAM-A monomers. Residues Glu(61) and Lys(63) of beta-strand C and Leu(72) of beta-strand C' in the dimer interface are required for efficient JAM-A engagement of strain type 3 Dearing sigma1. Mutation of neighboring residues alters the kinetics of the sigma1-JAM-A binding interaction. Prototype reovirus strains type 1 Lang and type 2 Jones share similar, although not identical, binding requirements with type 3 Dearing. These results indicate that reovirus engages JAM-A monomers via residues found mainly on beta-strands C and C' of the dimer interface and raise the possibility that the distinct disease phenotypes produced in mice following infection with different strains of reovirus are in part attributable to differences in contacts with JAM-A.

Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kappaB and blocked the expression of NF-kappaB-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-kappaB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIkappaBalpha. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-kappaB-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-kappaB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-kappaB-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-alpha, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-kappaB-directed expression of cellular genes that activate death signaling pathways in infected cells.

Apoptosis of uninfected bystander CD4+T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4+T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.