Reovirus Virions Research Articles

Reovirus Low-Density Particles Package Cellular RNA.

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and “genomeless” top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

Reovirus directly engages integrin to recruit clathrin for entry into host cells

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, β1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct β1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and β1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and β1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for β1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and β1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

Abstract 2169: Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis

Abstract Breast cancer remains the most diagnosed type of cancer and second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancer cases. TNBCs are characterized by lack of estrogen (ER) and progesterone (PR) receptors and growth factor receptor HER2/Neu on the cell surface. Therapies that target ER, PR, or HER2 are thus ineffective against TNBC. TNBC is primarily treated with cytotoxic chemotherapy or radiation therapy, which can cause significant damage to healthy cells and tissue. Therefore, there is need for therapies that selectively kill TNBC cells with reduced off-tumor effects. The concept of oncolytic virotherapy has existed since the early 1900s. However, oncolytic viruses have limited efficacy as single agents. Mammalian orthoreovirus (reovirus) is a non-enveloped, segmented double-stranded RNA virus with tropism for transformed cells. Reovirus is in Phase I - III clinical trials to test its oncolytic efficacy against a variety of cancers. Success has been limited and few trials have tested reovirus against breast cancer. In a high-throughput screen of small molecule inhibitors, we identified the topoisomerase II inhibitor doxorubicin hydrochloride (dox) as an enhancer of reovirus infectivity in the TNBC cell line MDA-MB-231. To better control dox delivery and enhance reovirus oncolytic potential, we chemically conjugated dox to reovirus virions (reo-dox). While reo-dox attachment to MDA-MB-231 cells is slightly impaired, viral replication is largely unaffected. Infection of MDA-MB-231 cells with reo-dox resulted in increased cytopathicity and faster induction of cell death than virus alone. MDA-MB-231 cells infected with reo-dox, but not virus alone, induced DNA double-strand breaks and activation of DNA damage response. Together, our findings show that reo-dox exhibits superior toxicity in TNBC cells than virus alone. Future studies will define the mechanism of enhanced cytopathicity of reo-dox and oncolytic efficacy of dox-conjugated reovirus in vivo. Delivery of small molecule inhibitors via conjugation to reovirus particles may provide an effective new method to directly target and kill cancer cells while minimizing toxicity to healthy cells and tissues. Citation Format: Jameson T. Berry, Bernardo A. Mainou. Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2169.

Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes.

Many host cellular signaling pathways were activated and exploited by virus infection for more efficient replication. The PI3K/Akt pathway has recently attracted considerable interest due to its role in regulating virus replication. This study demonstrated for the first time that the mammalian reovirus strains Masked Palm Civet/China/2004 (MPC/04) and Bat/China/2003 (B/03) can induce transient activation of the PI3K/Akt pathway early in infection in vitro. When UV-treated, both viruses activated PI3K/Akt signaling, indicating that the virus/receptor interaction was sufficient to activate PI3K/Akt. Reovirus virions can use both clathrin- and caveolae-mediated endocytosis, but only chlorpromazine, a specific inhibitor of clathrin-mediated endocytosis, or siRNA targeting clathrin suppressed Akt phosphorylation. We also identified the upstream molecules of the PI3K pathway. Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation. Blockage of PI3K/Akt activation increased virus RNA synthesis and viral yield. We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway. Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs. Overexpression of ISG15 and Viperin inhibited virus replication, and knockdown of either enhanced virus replication. Collectively, these results demonstrate that PI3K/Akt activated by mammalian reovirus serves as a pathway for sensing and then inhibiting virus replication/infection.

Diminished reovirus capsid stability alters disease pathogenesis and littermate transmission.

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.

Postvaccinal reovirus infection with high mortality in breeder chicks.

A broiler breeder flock was subcutaneously vaccinated at the hatchery with a live avian orthoreovirus (ARV) vaccine against viral arthritis. Chicks began to die at 3 days of age and postmortem examination revealed massive subcutaneous hemorrhages and edema on the dorsal aspect of the neck at the site of vaccination, a severe necrotic hepatitis, and pulmonary edema. Microscopically, the main lesion was a multifocal vacuolar degeneration and necrosis of randomly distributed small groups of hepatocytes with presence of apoptotic and multinucleated syncytial cells. Necrotic foci were also found in the lungs as well as a hemorrhagic, granulomatous, and heterophilic cellulitis and myositis of the neck and a generalized depletion and lymphocytolysis of lymphoid organs. At 8 days of age, birds also began to show hock swelling histologically characterized by a fibrinoleucocytic inflammation of the articulation and tendon sheaths, with hyperplasia of the synovial membrane, and lymphoplasmocytic infiltration. PCR and viral culture of livers were positive for ARV. Partial sequencing of the S1 gene from the virus isolate showed 99.2% to 99.8% homology with three vaccinal strains (ARV S1133, 1733, and 2408). Viral particles compatible with reovirus virions were observed at transmission electron microscopy. Investigation at the hatchery revealed that chicks were inadvertently administered an S1133 reovirus vaccine labeled for water administration in 10- to 17-week-old chickens. This human error is most likely the reason for this unusually severe viremic reovirus infection that affected this flock at such an early age.

The Nogo Receptor NgR1 Mediates Infection by Mammalian Reovirus

Neurotropic viruses, including mammalian reovirus, must disseminate from an initial site of replication to the central nervous system (CNS), often binding multiple receptors to facilitate systemic spread. Reovirus engages junctional adhesion molecule A (JAM-A) to disseminate hematogenously. However, JAM-A is dispensable for reovirus replication in the CNS. We demonstrate that reovirus binds Nogo receptor NgR1, a leucine-rich repeat protein expressed in the CNS, to infect neurons. Expression of NgR1 confers reovirus binding and infection of nonsusceptible cells. Incubating reovirus virions with soluble NgR1 neutralizes infectivity. Blocking NgR1 on transfected cells or primary cortical neurons abrogates reovirus infection. Concordantly, reovirus infection is ablated in primary cortical neurons derived from NgR1 null mice. Reovirus virions bind to soluble JAM-A and NgR1, while infectious disassembly intermediates (ISVPs) bind only to JAM-A. These results suggest that reovirus uses different capsid components to bind distinct cell-surface molecules, engaging independent receptors to facilitate spread and tropism.

A small molecule inhibits virion attachment to heparan sulfate- or sialic acid-containing glycans.

Primary attachment to cellular glycans is a critical entry step for most human viruses. Some viruses, such as herpes simplex virus type 1 (HSV-1) and hepatitis C virus (HCV), bind to heparan sulfate, whereas others, such as influenza A virus (IAV), bind to sialic acid. Receptor mimetics that interfere with these interactions are active against viruses that bind to either heparan sulfate or to sialic acid. However, no molecule that inhibits the attachment of viruses in both groups has yet been identified. Epigallocatechin gallate (EGCG), a green tea catechin, is active against many unrelated viruses, including several that bind to heparan sulfate or to sialic acid. We sought to identify the basis for the broad-spectrum activity of EGCG. Here, we show that EGCG inhibits the infectivity of a diverse group of enveloped and nonenveloped human viruses. EGCG acts directly on the virions, without affecting the fluidity or integrity of the virion envelopes. Instead, EGCG interacts with virion surface proteins to inhibit the attachment of HSV-1, HCV, IAV, vaccinia virus, adenovirus, reovirus, and vesicular stomatitis virus (VSV) virions. We further show that EGCG competes with heparan sulfate for binding of HSV-1 and HCV virions and with sialic acid for binding of IAV virions. Therefore, EGCG inhibits unrelated viruses by a common mechanism. Most importantly, we have identified EGCG as the first broad-spectrum attachment inhibitor. Our results open the possibility for the development of small molecule broad-spectrum antivirals targeting virion attachment. Importance: This study shows that it is possible to develop a small molecule antiviral or microbicide active against the two largest groups of human viruses: those that bind to glycosaminoglycans and those that bind to sialoglycans. This group includes the vast majority of human viruses, including herpes simplex viruses, cytomegalovirus, influenza virus, poxvirus, hepatitis C virus, HIV, and many others.

Reovirus Cell Entry Requires Functional Microtubules

ABSTRACTMammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells.

Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin-Darby canine kidney cells.

Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin-Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.

Reovirus Uses Multiple Endocytic Pathways for Cell Entry

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.

Transport to Late Endosomes Is Required for Efficient Reovirus Infection

Rab GTPases play an essential role in vesicular transport by coordinating the movement of various types of cargo from one cellular compartment to another. Individual Rab GTPases are distributed to specific organelles and thus serve as markers for discrete types of endocytic vesicles. Mammalian reovirus binds to cell surface glycans and junctional adhesion molecule-A (JAM-A) and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within organelles of the endocytic compartment, reovirus undergoes stepwise disassembly catalyzed by cathepsin proteases, which allows the disassembly intermediate to penetrate endosomal membranes and release the transcriptionally active viral core into the cytoplasm. The pathway used by reovirus to traverse the endocytic compartment is largely unknown. In this study, we found that reovirus particles traffic through early, late, and recycling endosomes during cell entry. After attachment to the cell surface, reovirus particles and JAM-A codistribute into each of these compartments. Transfection of cells with constitutively active and dominant-negative Rab GTPases that affect early and late endosome biogenesis and maturation influenced reovirus infectivity. In contrast, reovirus infectivity was not altered in cells expressing mutant Rab GTPases that affect recycling endosomes. Thus, reovirus virions localize to early, late, and recycling endosomes during entry into host cells, but only those that traverse early and late endosomes yield a productive infection.

Cell Entry-Associated Conformational Changes in Reovirus Particles Are Controlled by Host Protease Activity

Membrane penetration by reovirus requires successive formation of two cell entry intermediates, infectious subvirion particles (ISVPs) and ISVP*s. In vitro incubation of reovirus virions with high concentration of chymotrypsin (CHT) results in partial digestion of the viral outer capsid to form ISVPs. When virions are instead digested with low concentrations of chymotrypsin, the outer capsid is completely proteolyzed to form cores. We investigated the basis for the inverse relationship between CHT activity and protease susceptibility of the reovirus outer capsid. We report that core formation following low-concentration CHT digestion proceeds via formation of particles that contain a protease-sensitive form of the μ1C protein, a characteristic of ISVP*s. In addition, we found that both biochemical features and viral genetic requirements for ISVP* formation and core formation following low-concentration CHT digestion are identical, suggesting that core formation proceeds via a particle resembling ISVP*s. Furthermore, we determined that intermediates generated following low-concentration CHT digestion are distinct from ISVPs and convert to ISVP*-like particles much more readily than ISVPs. These results suggest that the activity of host proteases used to generate ISVPs can influence the efficiency with which the next step in reovirus cell entry, namely, ISVP-to-ISVP* conversion, occurs.

Src Kinase Mediates Productive Endocytic Sorting of Reovirus during Cell Entry

Reovirus cell entry is initiated by viral attachment to cell surface glycans and junctional adhesion molecule A. Following receptor engagement, reovirus is internalized into cells by receptor-mediated endocytosis using a process dependent on β1 integrin. Endocytosed virions undergo stepwise disassembly catalyzed by cathepsin proteases, followed by endosomal membrane penetration and delivery of transcriptionally active core particles into the cytoplasm. Cellular factors that mediate reovirus endocytosis are poorly defined. We found that both genistein, a broad-spectrum tyrosine kinase inhibitor, and PP2, a specific Src-family kinase inhibitor, diminish reovirus infectivity by blocking a cell entry step. Although neither inhibitor impedes internalization of reovirus virions, both inhibitors target virions to lysosomes. Reovirus colocalizes with Src during cell entry, and reovirus infection induces phosphorylation of Src at the activation residue, tyrosine 416. Diminished Src expression by RNA interference reduces reovirus infectivity, suggesting that Src is required for efficient reovirus entry. Collectively, these data provide evidence that Src kinase is an important mediator of signaling events that regulate the appropriate sorting of reovirus particles in the endocytic pathway for disassembly and cell entry.

Z-FA-FMK as a novel potent inhibitor of reovirus pathogenesis and oncolysis in vivo

Respiratory enteric orphan (reo)virus is a promising oncolytic viral candidate. Reoviral anticancer therapy is currently undergoing multiple clinical trials targeting various human cancers; however, there is no effective reoviral inhibitor that can be used to block unwanted reovirus replication during reoviral anticancer therapy. Studies were conducted with transformed or normal cells in vitro and in vivo to characterize viral replication in the presence or absence of chemical inhibitors. We have identified a protease inhibitor that is very effective in the inhibition of viral replication. The dipeptide benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (Z-FA-FMK) effectively inhibited reovirus replication in a susceptible host and cured cells of a persistent infection with reovirus in vitro. Electron microscopic analysis of Z-FA-FMK-treated cells revealed that internalized reovirus virions, retained in a perinuclear localization, no longer undergo further processing into viral factories following Z-FA-FMK treatment, suggesting that Z-FA-FMK specifically affects a reovirus virion maturation step. Animal studies showed that reovirus infection of Ras oncogenic tumours and host heart tissues is completely blocked by Z-FA-FMK treatment in severe combined immunodeficiency mice. Z-FA-FMK is a very effective viral inhibitor that can prevent reovirus replication in vitro and reovirus-mediated myocarditis, as well as reovirus-mediated oncolysis, in vivo. A potential application of this drug for inhibition of reovirus infection is suggested.

Transient infection of freshly isolated human colorectal tumor cells by reovirus T3D intermediate subviral particles

Reovirus T3D preferentially kills tumor cells expressing Ras oncogenes and has shown great promise as an anticancer agent in various preclinical tumor models. Here, we investigated whether reovirus can infect and kill tumor cell cultures and tissue fragments isolated from resected human colorectal tumors, and whether this was affected by the presence of endogenous oncogenic KRAS. Tissue fragments and single-cell populations isolated from human colorectal tumor biopsies were infected with reovirus virions or with intermediate subviral particles (ISVPs). Reovirus virions were capable of infecting neither single-cell tumor cell populations nor small fragments of intact viable tumor tissue. However, infection of tumor cells with ISVPs resulted in transient viral protein synthesis, irrespective of the presence of oncogenic KRAS, but this did not lead to the production of infectious virus particles, and tumor cell viability was largely unaffected. ISVPs failed to infect intact tissue fragments. Thermolysin treatment of tumor tissue liberated single cells from the tissue and allowed infection with ISVPs, but this did not result in the production of infectious virus particles. Immunohistochemistry on tissue microarrays showed that junction adhesion molecule 1, the major cellular reovirus receptor, was improperly localized in the cytoplasm of colorectal tumor cells and was expressed at very low levels in liver metastases. This may contribute to the observed resistance of tumor cells to reovirus T3D virions. We conclude that infection of human colorectal tumor cells by reovirus T3D requires processing of virions to ISVPs, but that oncolysis is prevented by a tumor cell response that aborts viral protein synthesis and the generation of infectious viral particles, irrespective of KRAS mutation status.

Proteolytic Disassembly Is a Critical Determinant for Reovirus Oncolysis

See page 1406Mammalian 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.

β1 Integrin Mediates Internalization of Mammalian Reovirus

Reovirus infection is initiated by interactions between the attachment protein sigma1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein lambda2, which serves as the structural base for sigma1, suggests that integrins mediate reovirus endocytosis. A beta1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a beta1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in beta1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing beta1. However, reovirus bound equivalently to cells that differed in levels of beta1 expression, suggesting that beta1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into beta1-deficient cells was substantially diminished in comparison to viral uptake into beta1-expressing cells. These data provide evidence that beta1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.

Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication.

We have utilized growth factors in in vitro and in vivo systems to examine the role of cellular proliferation in reovirus replication. In vitro, proliferating RIE-1 cells can be infected with whole reovirus virions, but are relatively resistant to infection once confluent (Go arrest). It has been shown that TGF-alpha, which signals through the EGF-receptor (EGF-R), is capable of dramatically increasing the number of RIE-1 cells entering the S-phase in the presence of additional serum factors. Stimulation of the EGF-R without serum results in minimal increases in cells entering the S-phase with a restriction in reovirus replication. Therefore, other factors in serum are essential for fully permissive infection. In vivo, we used metallothionein (MT) promoter/enhancer-TGF-alpha transgenic mice to study the effect of cytokine activation on reovirus type 1 infection. Virus replication decreased following oral infection in these transgenic mice at 1 month of age, concordant with increased mucin production. Titers of reovirus obtained from the livers of 1 year old transgenic mice were approximately 10-fold higher than titers obtained in control mice. Taken together, these data indicate that while growth factor activation ultimately leads to an increase in virus infectivity, other factors may be necessary for reovirus replication.

Cathepsin B Is Inhibited in Mutant Cells Selected during Persistent Reovirus Infection

Persistent reovirus infections of murine L929 (L) fibroblast cells select mutant (LX) cells that do not support proteolytic disassembly of reovirus virions within the endocytic pathway. To better understand the function and regulation of endocytic proteases, we conducted experiments to define the block to reovirus disassembly displayed by LX cells. In contrast to parental L cells, mutant LX cells harbor defects that interfere with the maturation and activity of cathepsin B and cathepsin L but not cathepsin H. The cDNAs encoding cathepsin B and cathepsin L in L cells are identical to those in LX cells, indicating that LX cells manifest an extrinsic block to the function of these enzymes. Mixed lysates of L cells and LX cells lack activity of both cathepsin B and cathepsin L, suggesting the presence of an inhibitor of cathepsin function in LX cells. A cathepsin B-green fluorescent protein (GFP) fusion protein expressed in L cells and purified by immunoprecipitation retains cathepsin B activity, whereas cathepsin B-GFP expressed in LX cells does not. However, activity of cathepsin B-GFP expressed in LX cells can be recovered by incubating the immunoprecipitate with L cell lysate followed by immunoprecipitation, providing further evidence that LX cells express a cathepsin inhibitor. Native-gel electrophoresis and gel filtration chromatography demonstrate that, in both cell lines, the double-chain form of cathepsin B is sequestered in a large molecular weight complex that renders this form of the enzyme inactive. Alteration of this sequestration complex appears to be responsible for inhibition of cathepsin B in LX cells. These findings suggest that cathepsins can be regulated within the endocytic pathway. Moreover, this regulation influences host cell susceptibility to intracellular pathogens.