Sequencing of serially passaged measles virus affirms its genomic stability and reveals a nonrandom distribution of consensus mutations.

Oncolytic virotherapy is an emerging treatment option for numerous cancers, with several virus families currently being evaluated in clinical trials. More specifically, vaccine-strain measles virus has arisen as a promising candidate for the treatment of different tumour types in several early clinical trials. Replicating viruses, and especially RNA viruses without proofreading polymerases, can rapidly adapt to varying environments by selecting quasispecies with advantageous genetic mutations. Subsequently, these genetic alterations could potentially weaken the safety profile of virotherapy. In this study, we demonstrate that, following an extended period of virus replication in producer or cancer cell lines, the quasispecies consensus sequence of vaccine strain-derived measles virus accrues a remarkably small number of mutations throughout the nonsegmented negative-stranded RNA genome. Interestingly, we detected a nonrandom distribution of genetic alterations within the genome, with an overall decreasing frequency of mutations from the 3' genome start to its 5' end. Comparing the serially passaged viruses to the parental virus on producer cells, we found that the acquired consensus mutations did not drastically change viral replication kinetics or cytolytic potency. Collectively, our data corroborate the genomic stability and excellent safety profile of oncolytic measles virus, thus supporting its continued development and clinical translation as a promising viro-immunotherapeutic.

Both type I and type III interferons are required to restrict measles virus growth in lung epithelial cells.

Measles virus (MeV) first infects immune cells in the respiratory tract of a human host, spreads to lymphoid organs throughout the body, and finally enters and grows in respiratory epithelial cells before being released and transmitted to the next host. Thus, efficient growth in respiratory epithelial cells is important for the person-to-person transmission of MeV. Upon viral entry, host cells detect viral nucleic acids and produce interferons (IFNs) to control viral growth. Type I (IFN-α/ß) and type III (IFN-λ) IFNs have largely common induction and signaling mechanisms and stimulate expression of similar target genes but utilize distinct receptors. To determine the relative contributions of type I and type III IFNs to the control of MeV growth in epithelial cells, we examined the growth of MeV and that of its mutants lacking either type I or type III IFN receptor in the human lung epithelial cell line H358. Our results revealed that both type I and type III IFNs are required to restrict MeV growth in H358 cells and that the induction of type III as well as type I IFNs was increased in the absence of the MeV nonstructural V protein.

Sphingolipids in early viral replication and innate immune activation.

In this review, we summarize the mechanisms by which sphingolipids modulate virus multiplication and the host innate immune response, using a number of host-virus systems as illustrative models. Sphingolipids exert diverse functions, both at the level of the viral life cycle and in the regulation of antiviral immune responses. Sphingolipids may influence viral replication in three ways: by serving as (co)receptors during viral entry, by modulating virus replication, and by shaping the antiviral immune response. Several studies have demonstrated that sphingosine kinases (SphK) and their product, sphingosine-1-phosphate (S1P), enhance the replication of influenza, measles, and hepatitis B virus (HBV). In contrast, ceramides, particularly S1P and SphK1, influence the expression of type I interferon (IFN-I) by modulating upstream antiviral signaling and enhancing dendritic cell maturation, differentiation, and positioning in tissue. The synthetic molecule α-galactosylceramide has also been shown to stimulate natural killer cell activation and interferon (IFN)-γ secretion. However, to date, clinical trials have failed to demonstrate any clinical benefit for sphingolipids in the treatment of cancer or HBV infection. Taken together, these findings show that sphingolipids play an important and underappreciated role in the control of virus replication and the innate immune response.

High titer oncolytic measles virus production process by integration of dielectric spectroscopy as online monitoring system.

Oncolytic viruses offer new hope to millions of patients with incurable cancer. One promising class of oncolytic viruses is Measles virus, but its broad administration to cancer patients is currently hampered by the inability to produce the large amounts of virus needed for treatment (1010 -1012 virus particles per dose). Measles virus is unstable, leading to very low virus titers during production. The time of infection and time of harvest are therefore critical parameters in a Measles virus production process, and their optimization requires an accurate online monitoring system. We integrated a probe based on dielectric spectroscopy (DS) into a stirred tank reactor to characterize the Measles virus production process in adherent growing Vero cells. We found that DS could be used to monitor cell adhesion on the microcarrier and that the optimal virus harvest time correlated with the global maximum permittivity signal. In 16 independent bioreactor runs, the maximum Measles virus titer was achieved approximately 40 hr after the permittivity maximum. Compared to an uncontrolled Measles virus production process, the integration of DS increased the maximum virus concentration by more than three orders of magnitude. This was sufficient to achieve an active Measles virus concentration of > 1010 TCID50 ml-1 .

Screening different host cell lines for the dynamic production of measles virus.

Measles virus (MV) has a natural affinity for cancer cells and oncolytic MV preparations have therefore been investigated in several clinical trials as a potential treatment for cancer. The main bottleneck in the administration of oncolytic MV to cancer patients is the production process, because very large doses of virus particles are required for each treatment. Here, we investigated the productivity of different host cells and found that a high infection efficiency did not necessarily result in high virus yields because virus release is also dependent on the host cell. As well as producing large numbers of active MV particles, host cells must perform well in dynamic cultivation systems. In screening experiments, the highest productivity was achieved by Vero and BJAB cells, but only the Vero cells maintained their high virus productivity when transferred to a stirred tank reactor. We used dielectric spectroscopy as an online monitoring system to control the infection and harvest times, which are known to be critical process parameters. The precise control of these parameters allowed us to achieve higher virus titers with Vero cells in a stirred tank reactor than in a static cultivation system based on T-flasks, with maximum titers of up to 1011 TCID50 ml-1 . © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:989-997, 2017.

Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells.

UNLABELLED: Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection, and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically, monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results obtained with free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small-pore (0.4-µm) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large-pore (3.0-µm) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large-pore membranes but not small-pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4-dependent manner. Thus, both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE. IMPORTANCE: Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-to-cell contact rather than on particle release and reentry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-to-cell contact rather than through cell-free particles. In addition, we sought to determine which immune cells transfer MeV infectivity to the human airway epithelium. Our studies are based on two types of human primary cells: (i) myeloid cells generated from donated blood and (ii) well-differentiated airway epithelial cells derived from donor lungs. We show that different types of myeloid cells, i.e., monocyte-derived macrophages and dendritic cells, transfer infection to airway epithelial cells. Furthermore, cell-to-cell contact is an important component of successful MeV transfer. Our studies elucidate a mechanism by which the most contagious human respiratory virus is delivered to the airway epithelium.

Measles Virus Genotype D Wild Strains Suppress Interferon-Stimulated Gene Expression More Potently than Laboratory Strains in SiHa Cells.

Changes in interferon (IFN)-stimulated gene (ISG) expression in cells infected with measles virus (MeV), four wild strains (belonging to different genotypes), and the laboratory strain Edmonston were examined. ISGs [MxA, 2'-5'-oligoadenylate synthetase, and interferon regulatory factor-1] were upregulated in an MeV-infection-induced manner and in an IFN-induced manner. In MeV-infected SiHa cell lines, the MeV infection-induced expression levels were in the order of A>H1>D8>D5>D3. On the other hand, all infected cell lines abolished type I and III IFN-induced ISG expression. However, partial type II IFN-mediated induction was observed in the MeV-infected cells. The wild strain of genotype D3 was the most potent inhibitor of MeV infection-induced and IFN-induced ISG expression and generated the highest titer of infectious viral particles. Edmonston triggered the highest levels of MeV infection-induced ISG expression in SiHa cells and produced the lowest titer of infectious particles. Expression of the viral C protein was associated with suppression of MeV infection-induced and type II IFN-induced ISG expression.

Adenovirus Type 7 Pneumonia in Children Who Died from Measles-Associated Pneumonia, Hanoi, Vietnam, 2014.

During a 2014 measles outbreak in Vietnam, postmortem pathologic examination of hospitalized children who died showed that adenovirus type 7 pneumonia was a contributory cause of death in children with measles-associated immune suppression. Adenovirus type 7 pneumonia should be recognized as a major cause of secondary infection after measles.

Sensitivity of human pleural mesothelioma to oncolytic measles virus depends on defects of the type I interferon response.

Attenuated measles virus (MV) is currently being evaluated as an oncolytic virus in clinical trials and could represent a new therapeutic approach for malignant pleural mesothelioma (MPM). Herein, we screened the sensitivity to MV infection and replication of twenty-two human MPM cell lines and some healthy primary cells. We show that MV replicates in fifteen of the twenty-two MPM cell lines. Despite overexpression of CD46 by a majority of MPM cell lines compared to healthy cells, we found that the sensitivity to MV replication did not correlate with this overexpression. We then evaluated the antiviral type I interferon (IFN) responses of MPM cell lines and healthy cells. We found that healthy cells and the seven insensitive MPM cell lines developed a type I IFN response in presence of the virus, thereby inhibiting replication. In contrast, eleven of the fifteen sensitive MPM cell lines were unable to develop a complete type I IFN response in presence of MV. Finally, we show that addition of type I IFN onto MV sensitive tumor cell lines inhibits replication. These results demonstrate that defects in type I IFN response are frequent in MPM and that MV takes advantage of these defects to exert oncolytic activity.

The Nectin-4/Afadin Protein Complex and Intercellular Membrane Pores Contribute to Rapid Spread of Measles Virus in Primary Human Airway Epithelia.

UNLABELLED: The discovery that measles virus (MV) uses the adherens junction protein nectin-4 as its epithelial receptor provides a new vantage point from which to characterize its rapid spread in the airway epithelium. We show here that in well-differentiated primary cultures of airway epithelial cells from human donors (HAE), MV infectious centers form rapidly and become larger than those of other respiratory pathogens: human respiratory syncytial virus, parainfluenza virus 5, and Sendai virus. While visible syncytia do not form after MV infection of HAE, the cytoplasm of an infected cell suddenly flows into an adjacent cell, as visualized through wild-type MV-expressed cytoplasmic green fluorescent protein (GFP). High-resolution video microscopy documents that GFP flows through openings that form on the lateral surfaces between columnar epithelial cells. To assess the relevance of the protein afadin, which connects nectin-4 to the actin cytoskeleton, we knocked down its mRNA. This resulted in more-limited infectious-center formation. We also generated a nectin-4 mutant without the afadin-binding site in its cytoplasmic tail. This mutant was less effective than wild-type human nectin-4 at promoting MV infection in primary cultures of porcine airway epithelia. Thus, in airway epithelial cells, MV spread requires the nectin-4/afadin complex and is based on cytoplasm transfer between columnar cells. Since the viral membrane fusion apparatus may open the passages that allow cytoplasm transfer, we refer to them as intercellular membrane pores. Virus-induced intercellular pores may contribute to extremely efficient measles contagion by promoting the rapid spread of the virus through the upper respiratory epithelium. IMPORTANCE: Measles virus (MV), while targeted for eradication, still causes about 120,000 deaths per year worldwide. The recent reemergence of measles in insufficiently vaccinated populations in Europe and North America reminds us that measles is extremely contagious, but the processes favoring its spread in the respiratory epithelium remain poorly defined. Here we characterize wild-type MV spread in well-differentiated primary cultures of human airway epithelial cells. We observed that viral infection promotes the flow of cytoplasmic contents from infected to proximal uninfected columnar epithelial cells. Cytoplasm flows through openings that form on the lateral surfaces. Infectious-center growth is facilitated by afadin, a protein connecting the adherens junction and the actin cytoskeleton. The viral fusion apparatus may open intercellular pores, and the cytoskeleton may stabilize them. Rapid homogenization of cytoplasmic contents in epithelial infectious centers may favor rapid spread and contribute to the extremely contagious nature of measles.

Success of measles virotherapy in ATL depends on type I interferon secretion and responsiveness.

Adult T cell leukemia/lymphoma (ATL) is a highly aggressive CD4+/CD25+ T-cell malignancy caused by human T cell lymphotropic virus type 1 (HTLV-1). Previous studies in the MET-1 cell/NOD/SCID mouse model of ATL demonstrated that MET-1 cells are very susceptible to measles virus (MV) oncolytic therapy. To further evaluate the potential of MV therapy in ATL, the susceptibility of several HTLV-1 transformed CD4+ T cell lines (MT-1, MT-2, MT-4 and C8166-45) as well as HTLV-1 negative CD4+ T cell lines (Jurkat and CCRF-CEM) to infection with MV was tested in vitro. All cell lines were permissive to MV infection and subsequent cell death, except MT-1 and CCRF-CEM cells which were susceptible and permissive to MV infection, but resistant to cell death. The resistance to MV-mediated cell death was associated with IFNß produced by MT-1 and CCRF-CEM cells. Inhibition of IFNß rendered MT-1 and CCRF-CEM cells susceptible to MV-mediated cell death. Cells susceptible to MV-induced cell death did not produce nor were responsive to IFNß. Upon infection with Newcastle Disease Virus (NDV), MT-1 and CCRF-CEM but not the susceptible cell lines up-regulated pSTAT-2. In vivo, treatment of tumors induced by MT-1 cell lines which produce IFNß demonstrated only small increases in mean survival time, while only two treatments prolonged mean survival time in mice with MET-1 tumors deficient in type I interferon production. These results indicate that type I interferon production is closely linked with the inability of tumor cells to respond to type I interferon. Screening of tumor cells for type I interferon could be a useful strategy to select candidate patients for MV virotherapy.

Faster replication and higher expression levels of viral glycoproteins give the vesicular stomatitis virus/measles virus hybrid VSV-FH a growth advantage over measles virus.

UNLABELLED: VSV-FH is a hybrid vesicular stomatitis virus (VSV) with a deletion of its G glycoprotein and encoding the measles virus (MV) fusion (F) and hemagglutinin (H) envelope glycoproteins. VSV-FH infects cells expressing MV receptors and is fusogenic and effective against myeloma xenografts in mice. We evaluated the fusogenic activities of MV and VSV-FH in relationship to the density of receptor on the target cell surface and the kinetics of F and H expression in infected cells. Using a panel of cells expressing increasing numbers of the MV receptor CD46, we evaluated syncytium size in MV- or VSV-FH-infected cells. VSV-FH is not fusogenic at low CD46 density but requires less CD46 for syncytium formation than MV. The size of each syncytium is larger in VSV-FH-infected cells at a specific CD46 density. While syncytium size reached a plateau and did not increase further in MV-infected CHO cells expressing ≥4,620 CD46 copies/cell, there was a corresponding increase in syncytium size with increases in CD46 levels in VSV-FH-infected CD46-expressing CHO (CHO-CD46) cells. Further analysis in VSV-FH-infected cell lines shows earlier and higher expression of F and H mRNAs and protein. However, VSV-FH cytotoxic activity was reduced by pretreatment of the cells with type I interferon. In contrast, the cytopathic effects are not affected in MV-infected cells. In summary, VSV-FH has significant advantages over MV as an oncolytic virus due to its higher viral yield, faster replication kinetics, and larger fusogenic capabilities but should be used in cancer types with defective interferon signaling pathways. IMPORTANCE: We studied the cytotoxic activity of a vesicular stomatitis/measles hybrid virus (VSV-FH), which is superior to that of measles virus (MV), in different cancer cell lines. We determined that viral RNA and protein were produced faster and in higher quantities in VSV-FH-infected cells. This resulted in the formation of larger syncytia, higher production of infectious particles, and a more potent cytopathic effect in permissive cells. Importantly, VSV-FH, similar to MV, can discriminate between low- and high-expressing CD46 cells, a phenotype important for cancer therapy as the virus will be able to preferentially infect cancer cells that overexpress CD46 over low-CD46-expressing normal cells.

Innate immune defense defines susceptibility of sarcoma cells to measles vaccine virus-based oncolysis.

The oncolytic potential of measles vaccine virus (MeV) has been demonstrated in several tumor entities. Here, we investigated the susceptibility of eight sarcoma cell lines to MeV-mediated oncolysis and found five to be susceptible, whereas three proved to be resistant. In the MeV-resistant cell lines, we often observed an inhibition of viral replication along with a strong upregulation of the intracellular virus-sensing molecule RIG-I and of the interferon (IFN)-stimulated gene IFIT1. Not only expression of IFIT1 but also phosphorylation of IFN-stimulated Stat1 took place rapidly and were found to be persistent over time. In contrast, susceptible cell lines showed a much weaker, delayed, or completely missing expression of IFIT1 as well as a delayed or only transient phosphorylation of Stat1, whereas exogenic stimulation with beta interferon (IFN-ß) resulted in a comparable profound activation of Stat1 and expression of IFIT1 in all cell lines. Pretreatment with IFN-ß rendered three of the susceptible cell lines more resistant to MeV-mediated oncolysis. These data suggest that differences in the innate immune defense often account for different degrees of susceptibility of sarcoma cell lines to MeV-mediated oncolysis. From a therapeutic perspective, we were able to overcome resistance to MeV by increasing the multiplicity of infection (MOI) and by addition of the prodrug 5-fluorocytosine (FC), thereby exploiting the suicide gene function of virotherapeutic vector MeV-SCD armed with the SCD fusion protein, which consists of yeast cytosine deaminase and yeast uracil phosphoribosyltransferase.

The innate antiviral factor APOBEC3G targets replication of measles, mumps and respiratory syncytial viruses.

The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adeno-associated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1-2 logs (90-99 %) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50-70 %, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (C→U) or guanine to adenine (G→A) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, A→G and U→C transitions, with preference for next neighbour-nucleotides U = A>C>G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells.

Manufacture of measles viruses.

Measles viruses have shown potent oncolytic activity as a therapeutic against a variety of human cancers in animal models and are currently being tested in clinical trials in patients. In contrast to using measles virus as a vaccine, oncolytic activity depends on high concentrations of infectious virus. For use in humans, the high-titer measles virus preparations must also be purified to remove significant levels of cellular proteins and nucleic acid resulting from the cytolytic products of measles virus replication and release. Pleomorphic measles virus must be treated as >1-µm particles that are extremely shear sensitive to maximize recoveries and retain infectivity. Therefore, to maximize the recovery of sterile, high titer infectious measles viruses, the entire production and purification process must be done using gentle conditions and aseptic processing. Here we describe a procedure applicable to the production of small (a few liters) to large (50-60 L) batches of measles virus amplified in Vero cells adapted to serum-free growth. Cell culture supernatant containing the measles virus is clarified by filtration to remove intact Vero cells and other debris, and then treated with Benzonase(®) in the presence of magnesium chloride to digest contaminating nucleic acid. The measles virus in the treated cell culture supernatant is then concentrated and purified using tangential flow filtration (TFF) and diafiltration. The concentrated and diafiltered measles virus is passed through a final clarifying filter prior to final vialing and storage at <-65°C. An infectivity assay to quantify infectious measles virus concentration based on the TCID(50) method is also described. This procedure can be readily adapted to the production and purification of measles viruses using good manufacturing practices (GMP).

Amino Acid substitutions in matrix, fusion and hemagglutinin proteins of wild measles virus for adaptation to vero cells.

BACKGROUND: Wild-type measles virus (MV) is isolated in B95a but not in Vero cells. Through an adaptation process of wild-type MV to Vero cells, several amino acid substitutions were reported. METHODS: Six strains were adapted to Vero cells and membrane (M), fusion (F) and hemagglutinin (H) genes were sequenced. Cell fusion was assessed and recombinant MVs were constructed, having wild-type H or M gene with or without mutations. RESULTS: No F gene substitution was noted. Amino-acid substitutions at positions 481 from Asn to Tyr (N481Y) and 546 from Ser to Gly (S546G) were observed in the H protein. Glu at position 89 of the M protein was substituted for Gly (E89G) and two mutations were noted at positions 62 (S62R) and 83 (S83P) in M protein. Recombinant viruses with mutation(s) detected in Vero-adapted strains induced a cytopathic effect and grew well in Vero cells, but those with the wild type did not. Recombinant viruses with mutation(s) demonstrated lower viral growth in B95a cells. CONCLUSIONS: Substitutions of E89G, S62R and S83P of the M protein were newly observed through adaptation to Vero cells, besides the mutations described in previous reports, with varying adaptation for each strain.

Use of attenuated paramyxoviruses for cancer therapy.

Paramyxoviruses, measles virus (MV), mumps virus (MuV) and Newcastle disease virus (NDV), are well known for causing measles and mumps in humans and Newcastle disease in birds. These viruses have been tamed (attenuated) and successfully used as vaccines to immunize their hosts. Remarkably, pathogenic MuV and vaccine strains of MuV, MV and NDV efficiently infect and kill cancer cells and are consequently being investigated as novel cancer therapies (oncolytic virotherapy). Phase I/II clinical trials have shown promise but treatment efficacy needs to be enhanced. Technologies being developed to increase treatment efficacy include: virotherapy in combination with immunosuppressive drugs (cyclophosphamide); retargeting of viruses to specific tumor types or tumor vasculature; using infected cell carriers to protect and deliver the virus to tumors; and genetic manipulation of the virus to increase viral spread and/or express transgenes during viral replication. Transgenes have enabled noninvasive imaging or tracking of viral gene expression and enhancement of tumor destruction.

Specific CD8(+) T-lymphocytes control dissemination of measles virus.

Measles continues to be an important cause of childhood mortality in developing countries. Measles virus (MV) is lymphotropic and infects high percentages of B- and T-lymphocytes in lymphoid tissues. Cellular immunity is considered crucial for viral clearance; however, MV-specific T-lymphocytes generated during primary infection also constitute a potential target for MV infection. We therefore aimed to identify T-lymphocyte subsets that can clear MV infection without becoming infected. To this end, we infected human EBV transformed B-lymphoblastic cell lines (B-LCL) with a recombinant MV strain expressing enhanced GFP, and co-cultured these with non-infected B-LCL resulting in rapid viral spread. MV-specific CD8(+) T-cell clones efficiently suppressed MV dissemination in autologous and HLA-matched, but not in HLA-mismatched B-LCL. In contrast, CD4(+) T-cell clones could not control MV dissemination but became a target for MV infection themselves. Furthermore, PBMC collected 6-9 months after acute measles and stimulated with autologous MV-infected B-LCL also efficiently suppressed MV dissemination; this was mediated by the fraction containing CD8(+) T-lymphocytes. In conclusion, we have developed a powerful tool to study cellular immunity against measles, and demonstrate that control of MV dissemination is mediated by virus-specific CD8(+) rather than by CD4(+) T-lymphocytes.

RNA-specific adenosine deaminase ADAR1 suppresses measles virus-induced apoptosis and activation of protein kinase PKR.

ADAR1 (adenosine deaminase acting on RNA) catalyzes the conversion of adenosine to inosine, a process known as A-to-I editing. Extensive A-to-I editing has been described in viral RNAs isolated from the brains of patients persistently infected with measles virus, although the precise role of ADAR during measles virus infection remains unknown. We generated human HeLa cells stably deficient in ADAR1 ("ADAR1(kd) cells") through short hairpin RNA-mediated knockdown, and using these cells, we tested the effect of ADAR1 deficiency on measles virus (MVvac strain) growth and virus-induced cell death. We found that the growth of mutant viruses lacking expression of the viral accessory proteins V and C (V(ko) and C(ko), respectively) was decreased in ADAR1-deficient cells compared with ADAR1-sufficient cells. In addition, apoptosis was enhanced in ADAR1-deficient cells following infection with wild type and V(ko) virus but not following infection with C(ko) virus or treatment with tumor necrosis factor-alpha or staurosporine. Furthermore, in C(ko)-infected ADAR1-sufficient cells when ADAR1 did not protect against apoptosis, caspase cleavage of the ADAR1 p150 protein was detected. Finally, enhanced apoptosis in ADAR1(kd) cells following infection with wild type and V(ko) virus correlated with enhanced activation of PKR kinase and interferon regulatory factor IRF-3. Taken together, these results demonstrate that ADAR1 is a proviral, antiapoptotic host factor in the context of measles virus infection and suggest that the antiapoptotic activity of ADAR1 is achieved through suppression of activation of proapoptotic and double-stranded RNA-dependent activities, as exemplified by PKR and IRF-3.

Previously unrecognized amino acid substitutions in the hemagglutinin and fusion proteins of measles virus modulate cell-cell fusion, hemadsorption, virus growth, and penetration rate.

Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.