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As countries and regions move toward measles elimination, extended sequence window including noncoding region located between the matrix and fusion protein genes (M − F NCR) was considered to be used in molecular surveillance. The molecular resolution of M − F NCR was evaluated with 192 genotype H1 strains circulating during 2011–2018 in China. Phylogenetic analyses of the N450 and M − F NCR targets indicated that both two targets could confirm epi-linked outbreak, while M − F NCR target could further improve resolution of the molecular characterization: (1) it could differentiate the strains with identical N450 circulated in one county within one month of disease onset; (2) different transmission chains could be distinguished for strains with identical N450; (3) better spatial-temporal consistency with topology could be provided among sporadic cases with inconsistent N450. Accordingly, M − F NCR could be used to complement the information from N450 to address the specific questions in tracking the virus transmission chains. • The limited genetic variation of N450 hampers its effective use in surveillance, especially during the elimination stage. • Compared with N450, M − F NCR even provided higher resolution in virus strains from outbreak and sporadic cases. • M − F NCR could be used to complement the information from N450 in molecular surveillance for virus transmission tracking. [ABSTRACT FROM AUTHOR]

Subacute sclerosing panencephalitis (SSPE) is a rare progressive neurodegenerative disease caused by measles virus variants (SSPE viruses) that results in eventual death. Amino acid substitution(s) in the viral fusion (F) protein are key for viral propagation in the brain in a cell-to-cell manner, a specific trait of SSPE viruses, leading to neuropathogenicity. In this study, we passaged an SSPE virus in cultured human neuronal cells and isolated an adapted virus that propagated more efficiently in neuronal cells and exhibited increased cell-to-cell fusion. Contrary to our expectation, the virus harbored mutations in the large protein, a viral RNA-dependent RNA polymerase, and in the phosphoprotein, its co-factor, rather than in the F protein. Our results imply that upregulated RNA polymerase activity, which increases F protein expression and cell-to-cell fusion, could be a viral factor that provides a growth advantage and contributes to the adaptation of SSPE viruses to neuronal cells. • An SSPE virus further adapted to neuronal cells by enhancing cell-to-cell infection. • Increase of fusogenicity of the F protein was not involved in the adaptation. • Upregulation of viral RNA polymerase (RdRp) promoted cell-to-cell infection. • Viral RdRp is a factor to cause adaptation of SSPE virus in neuronal cells. [ABSTRACT FROM AUTHOR]

The Peste des petits ruminant virus (PPRV) is a member of the Paramyxoviridae family and is classified into the genus Measles virus. PPRV predominantly infects small ruminants, leading to mortality rates of nearly 100%, which have caused significant economic losses in developing countries. Host proteins are important in virus replication, but the PPRV nucleocapsid (N) protein-host interacting partners for regulating PPRV replication remain unclear. The present study confirmed the interaction between PPRV-N and the host protein vimentin by co-immunoprecipitation and co-localization experiments. Overexpression of vimentin suppressed PPRV replication, whereas vimentin knockdown had the opposite effect. Mechanistically, N was subjected to degradation via the ubiquitin/proteasome pathway, where vimentin recruits the E3 ubiquitin ligase NEDD4L to fulfill N -ubiquitination, resulting in the degradation of the N protein. These findings suggest that the host protein vimentin and E3 ubiquitin ligase NEDD4L have an anti -PPRV effect. • Host Vimentin protein interacts with PPRV N protein. • Vimentin protein inhibits PPRV replication. • Vimentin recruits the E3 ubiquitin ligase NEDD4L to increase degradation of the PPRV N protein. [ABSTRACT FROM AUTHOR]

BST2/tetherin is a transmembrane protein with antiviral activity; it is synthesized following exposure to interferons, and restricts the release of budding virus particles by tethering them to the host cell membrane. We previously showed that BST2 is induced in primary neurons following measles virus (MV) infection or type I interferon; however, BST2 was dispensable for protection against challenge with neuron-restricted MV. Here, we define the contribution of BST-2 in neuronal MV infection. Surprisingly, and in contrast to its antiviral role in non-neuronal cells, murine BST2 promotes MV infection in brains of permissive mice and in primary neuron cultures. Moreover, BST2 expression was predominantly observed in the non-synaptic fraction of purified neurons. These studies highlight a cell-type dependent role of a well-characterized antiviral protein in enhancing neuronal infection. • BST2 promotes neuronal measles virus infection in primary neuronal cultures. • BST2 promotes neuronal measles virus infection in the brain. • BST2 is increased away from the neuronal synapse. [ABSTRACT FROM AUTHOR]

Abstract Measles virus has been successfully attenuated on chicken embryo cells to obtain a highly efficient and safe live attenuated vaccine, administered thus far to billions of children. Measles virus attenuation has long been described to involve a modification of cellular tropism with the use of human CD46 ubiquitous receptor. Nevertheless, the use of this receptor in vivo is not obvious. In this study we use four different mouse models to decipher the respective part of hCD46 receptor and type-I interferon response in measles host restriction. We observed that only type-I interferon restricts viral replication of attenuated MV Schwarz strain in mice, independently of the presence of hCD46 receptor. By comparing measles virus immunogenicity in the different models, we confirmed that there was no impact on the absence of this receptor on the immune response. Therefore, we propose to simplify the mouse model. [ABSTRACT FROM AUTHOR]

Cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to occur, making it one of the WHO´s targets for accelerated vaccine development. One vaccine candidate is based on live-attenuated measles virus (MV) vaccine encoding the MERS-CoV spike glycoprotein (MERS-S). MV vac2 -MERS-S(H) induces robust humoral and cellular immunity against MERS-S mediating protection. Here, the induction and nature of immunity after vaccination with MV vac2 -MERS-S(H) or novel MV vac2 -MERS-N were further characterized. We focused on the necessity for vector replication and the nature of induced T cells, since functional CD8 + T cells contribute importantly to clearance of MERS-CoV. While no immunity against MERS-CoV or MV was detected in MV-susceptible mice after immunization with UV-inactivated virus, replication-competent MV vac2 -MERS-S(H) triggered robust neutralizing antibody titers also in adult mice. Furthermore, a significant fraction of MERS CoV-specific CD8 + T cells and MV-specific CD4 + T cells simultaneously expressing IFN-γ and TNF-α were induced, revealing that MV vac2 -MERS-S(H) induces multifunctional cellular immunity. [ABSTRACT FROM AUTHOR]

The live-attenuated measles virus (MV) vaccine based on the Hu191 strain has played a significant role in controlling measles in China. However, it has considerable adverse effects that may cause public health burden. We hypothesize that the safety and efficacy of MV vaccine can be improved by altering the S-adenosylmethionine (SAM) binding site in the conserved region VI of the large polymerase protein. To test this hypothesis, we established an efficient reverse genetics system for the rMV-Hu191 strain and generated two recombinant MV-Hu191 carrying mutations in the SAM binding site. These two mutants grew to high titer in Vero cells, were genetically stable, and were significantly more attenuated in vitro and in vivo compared to the parental rMV-Hu191 vaccine strain. Importantly, both MV-Hu191 mutants triggered a higher neutralizing antibody than rMV-Hu191 vaccine and provided complete protection against MV challenge. These results demonstrate its potential for an improved MV vaccine candidate. [ABSTRACT FROM AUTHOR]

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE). We previously reported that the F gene of the SSPE Osaka-2 strain is the major determinant of MV neurovirulence. Because the sites and extents of mutations differ among SSPE strains, it is necessary to determine the mutations responsible for the SSPE-specific phenotypes of individual viral strain. In this study, recombinant viruses containing the envelope-associated genes from the SSPE Osaka-1 strain were generated in the IC323 wild-type MV background. Hamsters inoculated with MV containing the H gene of the Osaka-1 strain displayed hyperactivity and seizures, but usually recovered and survived. Hamsters inoculated with MV containing the F gene of the Osaka-1 strain displayed severe neurologic signs and died. Amino acid substitutions in the heptad repeat A and C regions of the F protein, including a methionine-to-valine substitution at amino acid 94, play major roles in neurovirulence. [ABSTRACT FROM AUTHOR]

Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. [ABSTRACT FROM AUTHOR]

Measles virus (MV) is the causative agent of measles and its neurological complications, subacute sclerosing panencephalitis (SSPE) and measles inclusion body encephalitis (MIBE). Biased hypermutation in the M gene is a characteristic feature of SSPE and MIBE. To determine whether the M gene is the preferred target of hypermutation, an additional transcriptional unit containing a humanized Renilla reniformis green fluorescent protein (hrGFP) gene was introduced into the IC323 MV genome, and nude mice were inoculated intracerebrally with the virus. Biased hypermutation occurred in the M gene and also in the hrGFP gene when it was inserted between the leader and the N gene, but not between the H and L gene. These results indicate that biased hypermutation is usually found in a gene whose function is not essential for viral proliferation in the brain and that the location of a gene in the MV genome can affect its mutational frequency. [ABSTRACT FROM AUTHOR]

Abstract: Measles virus (MV) deficient in C protein (C ko) expression efficiently induces both stress granules (SG) and interferon (IFNβ), whereas isogenic wild-type (WT) and V mutant (V ko) viruses do not. We therefore examined the effect of IFNβ pretreatment on SG formation, and the roles played by the IFN-inducible double-stranded (ds) RNA-dependent protein kinase (PKR) and dsRNA adenosine deaminase (ADAR1). SG formation in ADAR1-sufficient cells infected with WT or V ko mutant virus was enhanced by IFN treatment and was PKR-dependent. SG formation in C ko virus-infected cells was already high without IFN treatment and was not further enhanced by IFN. IFN treatment alone, in the absence of infection, induced SG formation in ADAR1-deficient but not ADAR1-sufficient cells. Type I IFN-induced enhancement in SG formation occurred by a canonical IFN signaling response dependent upon STAT1 and STAT2. These results further establish ADAR1 as a suppressor of the interferon and SG innate immune responses. [Copyright &y& Elsevier]

Abstract: The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. [Copyright &y& Elsevier]

Abstract: Measles virus (MV) manipulates host factors to facilitate virus replication. Sphingosine kinase (SK) is an enzyme catalyzing the formation of sphingosine 1-phosphate and modulates multiple cellular processes including the host defense system. Here, we determined the role of SK1 in MV replication. Overexpression of SK1 enhanced MV replication. In contrast, inhibition of SK impaired viral protein expression and infectious virus production from cells expressing MV receptor, SLAM or Nectin-4. The inhibition of virus replication was observed when the cells were infected by vaccine strain or wild type MV or V/C gene-deficient MV. Importantly, SK inhibition suppressed MV-induced activation of NF-κB. The inhibitors specific to NF-κB signal pathway repressed the synthesis of MV proteins, revealing the importance of NF-κB activation for efficient MV replication. Therefore, SK inhibition restricts MV replication and modulates the NF-κB signal pathway, demonstrating that SK is a cellular factor critical for MV replication. [Copyright &y& Elsevier]

Abstract: The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainly to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. [Copyright &y& Elsevier]

Abstract: The protein kinase regulated by RNA (PKR) enhances both activation of mitogen-activated protein kinases and the induction of interferon beta (IFN-β) by measles virus defective in C-protein expression (Cko). Here we used complementation of human cell lines stably deficient in PKR (PKRkd) to probe the basis of these PKR-mediated responses. We found that PKRkd HeLa and amnion U cell lines were defective for virus-mediated activation of IFN induction signaling components compared to PKR-sufficient control cells. Complementation of PKRkd cells with wildtype PKR, but not with PKR mutants defective in either catalytic activity or dsRNA-binding activity, restored JNK, p38 and ATF-2 phosphorylation and enhanced IFN-β induction following infection. By contrast to mammalian PKR, the Z-DNA binding domain-containing fish homologue of PKR, PKZ, lacked the capacity to enhance Cko virus-mediated IFN-β induction. Furthermore, inhibition of virus growth was observed with Cko-infected PKRkd cells complemented with PKR but not with PKZ. [Copyright &y& Elsevier]

Abstract: Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-α/β and γ-antagonist as strong as P gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways. [Copyright &y& Elsevier]

Abstract: We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes. [Copyright &y& Elsevier]

Abstract: Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression of C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-γ signaling pathway via inhibition of phosphorylated STAT1 dimerization. [Copyright &y& Elsevier]

Abstract: The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential. [Copyright &y& Elsevier]

Abstract: Sendai virus (SeV) HN protein is dispensable for virus particle production. HN incorporation into virions strictly depends on a cytoplasmic domain SYWST motif. HNAFYKD, with SYWST replaced with the analogous sequence of measles virus (MeV) H (AFYKD), is not incorporated in virus particles produced by LLCMK2 cells, although it is normally expressed at the plasma membrane. Unlike HNSYWST, HNAFYKD is not internalized to late endosomes, raising the possibility that HN internalization is required for uptake into virus particles. Various mosaic MeV-H containing increasing amounts of the SeV-HN all failed to be taken up in SeV virions. However, when co-expressed with HNAFYKD these MeV-H chimera induced HNAFYKD uptake into virions showing that internalization is not a prerequisite for HN uptake into particles. We propose that HN incorporation in virus particles requires first neutralization by HN of a putative inhibitor of infectious particle formation. [ABSTRACT FROM AUTHOR]

Abstract: The morbilliviruses are a closely related genus which are very similar in their sequences and share a common receptor, but nevertheless show significant restriction in the host species in which they cause disease. One contribution to this restriction might be the nature of the hosts'' responses to infection. We have used microarrays to study the changes in the transcriptome of bovine dendritic cells after infection with wild-type (pathogenic) and vaccine (apathogenic) strains of rinderpest virus (RPV), a bovine pathogen, and a wild-type isolate of measles virus (MV), a morbillivirus that causes disease only in humans and some other primates. We found that, as previously observed in human cells, MV induces a rapid interferon response, while that induced by RPV was delayed and much reduced in magnitude. Pathogenic and apathogenic RPV also showed significant differences, with the latter inducing a slightly higher interferon response as well as significant effects on transcription of genes involved in cell cycle regulation. [Copyright &y& Elsevier]

Abstract: Fusion promotion by measles virus (MV) depends on an interaction between the hemagglutinin (H) and fusion (F) glycoproteins. Amino acid substitutions in MV H that drastically reduce hemagglutinating activity result in an increase in the amount of H (primarily the 74 kDa isoform) detectable in a complex with F at the cell surface. This is in direct contrast to the loss of the ability to detect a complex between the fusion protein of Newcastle disease virus and most attachment proteins that lack receptor binding activity. These opposing results provide support for the existence of different mechanisms for the regulation of fusion by these two paramyxoviruses. [Copyright &y& Elsevier]

Abstract: The V protein of measles virus (MV-V) is a potent inhibitor of IFN-α/β signaling pathway. We previously reported that when physically dissociated, the N-terminal and C-terminal regions of MV-V (PNT and VCT, respectively) could independently impair signal transduction. The PNT region inhibited IFN-α/β signaling by interacting with at least two components of this pathway: Jak1 and STAT1. Here we report a direct interaction between the VCT of MV-V and STAT2, a third component of IFN-α/β transduction machinery. This interaction with STAT2 is carried by the cysteine-constrained peptide of 49 amino acids localized in the VCT region, and is essential to the inhibition of IFN-α/β signaling. In parallel, we also mapped STAT1 binding site in the PNT region and identified a minimal peptide of only 11 amino acids. IFN-α/β signaling was impaired in human cells treated with this MV-V peptide fused to a cell-penetrating sequence. Finally, we show that signaling downstream of IFN-λ, a recently identified cytokine that also relies on STAT1, STAT2 and Jak1 to transduce, is blocked by MV-V. Altogether, our results illustrate how a single viral protein has evolved to achieve a robust inhibition of the antiviral response by interacting with several signaling molecules. [Copyright &y& Elsevier]

Abstract: Measles virus (MV) causes various responses including the induction of immune responses, transient immunosuppression and establishment of long-lasting immunity. To obtain a comprehensive view of the effects of MV infection on target cells, DNA microarray analyses of two different cell-types were performed. An epithelial (293SLAM; a 293 cell line stably expressing SLAM) and lymphoid (COBL-a) cell line were inoculated with purified wild-type MV. Microarray analyses revealed significant differences in the regulation of cellular gene expression between these two different cells. In 293SLAM cells, upregulation of genes involved in the antiviral response was rapidly induced; in the later stages of infection, this was followed by regulation of many genes across a broad range of functional categories. On the other hand, in COBL-a cells, only a limited set of gene expression profiles was modulated after MV infection. Since it was reported that V protein of MV inhibited the IFN signaling pathway, we performed a microarray analysis using V knockout MV to evaluate V protein''s effect on cellular gene expression. The V knockout MV displayed a similar profile to that of parental MV. In particular, in COBL-a cells infected with the virus, no alteration of cellular gene expression, including IFN signaling, was observed. Furthermore, IFN signaling analyzed in vitro was completely suppressed by MV infection in the COBL-a cells. These results reveal that MV induces different cellular responses in a cell-type specific manner. Microarray analyses will provide us useful information about potential mechanisms of MV pathogenesis. [Copyright &y& Elsevier]

Abstract: The C and V proteins of the measles virus (MV) have been shown to block the signaling of type I and II interferon (IFN-α/β and IFN-γ). The relative contribution of the C and V proteins to the inhibition of IFN signaling and the extent to which this activity differs in attenuated or wild-type strains of MV remains undefined. This study presents a comparison of the IFN-antagonist activities of C and V proteins from four attenuated and two wild-type strains of MV. The V proteins were more potent inhibitors of IFN-inducible reporter gene expression than the C proteins, and this effect was unrelated to whether the protein originated from an attenuated or wild-type strain. The results also demonstrated the importance of the tyrosine at position 110 in the inhibition of IFN-α/β and IFN-γ signaling by the V protein, and identified a non-recombinant MV expressing a V protein that was impaired due to a mutation at this residue. [Copyright &y& Elsevier]

Abstract: Viruses have evolved various strategies to escape the antiviral activity of type I interferons (IFN-α/β). For measles virus, this function is carried by the polycistronic gene P that encodes, by an unusual editing strategy, for the phosphoprotein P and the virulence factor V (MV-V). MV-V prevents STAT1 nuclear translocation by either sequestration or phosphorylation inhibition, thereby blocking IFN-α/β pathway. We show that both the N- and C-terminal domains of MV-V (PNT and VCT) contribute to the inhibition of IFN-α/β signaling. Using the two-hybrid system and co-affinity purification experiments, we identified STAT1 and Jak1 as interactors of MV-V and demonstrate that MV-V can block the direct phosphorylation of STAT1 by Jak1. A deleterious mutation within the PNT domain of MV-V (Y110H) impaired its ability to interact and block STAT1 phosphorylation. Thus, MV-V interacts with at least two components of IFN-α/β receptor complex to block downstream signaling. [Copyright &y& Elsevier]

Abstract: The association between HLA homozygosity and measles-specific Th1 (IFN-γ, IL-2 and IL-12p40) and Th2 (IL-4 and IL-10) cytokine responses were assessed in a group of 339 healthy schoolchildren 12–18 years of age previously immunized with two doses of live-attenuated measles virus vaccine. No associations were observed between class I HLA homozygosity and measles-specific cytokine levels. Children who were homozygous at the class II DRB1, DQA1, DPA1 and DPB1 loci had higher median IFN-γ secretion levels compared with children who were heterozygous for DRB1 (77.7 vs. 39.5 pg/ml, p =0.05), DQA1 (60.9 vs. 36.6 pg/ml, p =0.03), DPA1 (46.1 vs. 27.1 pg/ml, p =0.01) and DPB1 (61.5 vs. 36.0 pg/ml, p =0.01) loci, respectively. Homozygosity at increasing numbers of HLA loci ( >=4) was associated with increased IFN-γ secretion levels (test for trend p-value=0.01). Our results suggest that HLA homozygosity showed no disadvantage for measles-specific cytokine responses and instead was associated with increased IFN-γ levels. [Copyright &y& Elsevier]

Abstract: Measles virus (MV), a morbillivirus that remains a significant human pathogen, can infect the central nervous system, resulting in rare but often fatal diseases, such as subacute sclerosing panencephalitis. Previous work demonstrated that MV was transmitted trans-synaptically and that, while a cellular receptor for the hemagglutinin (H) protein was required for MV entry, it was dispensable for subsequent cell-to-cell spread. Here, we explored what role the other envelope protein, fusion (F), played in trans-synaptic transport. We made the following observations: (1) MV-F expression in infected neurons was similar to that seen in infected fibroblasts; (2) fusion inhibitory peptide (FIP), an inhibitor of MV fusion, prevented both infection and spread in primary neurons; (3) Substance P, a neurotransmitter with the same active site as FIP, also blocked neuronal MV spread; and (4) both genetic deletion and pharmacological inhibition of the Substance P receptor, neurokinin-1 (NK-1), reduced infection of susceptible mice. Together, these data implicate a role for NK-1 in MV CNS infection and spread, perhaps serving as an MV-F receptor or co-receptor on neurons. [Copyright &y& Elsevier]

Abstract: A major cause of the high morbidity and mortality associated with measles infection is attributed to virus-mediated immunosuppression. In this report, we present evidence for a novel strategy of immunosuppression by the measles virus. We observed a marked suppression of lipopolysaccharide (LPS)-induced IL-8, RANTES, TNF-α and IL-6 production and NF-κB activation in human monocytic cell lines persistently infected with measles virus. This effect was not observed in human epithelial cells lines persistently infected with measles virus. There were no significant differences in expression levels of Toll-like receptors (TLRs) and their associated molecules, or other intracellular signaling molecules of the NF-κB signaling pathway in measles-virus-infected monocytic cells compared to uninfected cells. Infected monocytic cells exhibited decreased LPS-induced DNA binding of NF-κB and phosphorylation of JNK, namely activation of transcription factors NF-κB and AP-1. NF-κB was constitutively activated in human epithelial cells persistently infected with measles virus, and LPS treatment resulted in further activation. The cell-type-specific suppression of NF-κB activation represents a potential strategy of escape from the host immune system by measles virus via induced immunological silencing in infected cells. [Copyright &y& Elsevier]

Abstract: The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein. [Copyright &y& Elsevier]

Abstract: Two hallmarks of measles virus (MV) infection are the ability of the virus to cause immunosuppression and the resultant enhanced susceptibility of the infected host to microbial insults. We investigated the effect of MV infection on the ability of dendritic cells (DCs) to induce IL-12 via toll-like receptor (TLR) signaling. When infected with MV, transgenic mice which expressed human SLAM receptor on their DCs were defective in the selective synthesis of IL-12 in DCs in response to stimulation of TLR4 signaling, but not to engagements of TLR2, 3, 7 or 9. MV suppressed TLR4-mediated IL-12 induction in DCs even in the presence of co-stimulation with another ligand for TLR2, 3, 7, or 9. While MV V and C proteins were not responsible for IL-12 inhibition, interaction of MV hemagglutinin with human SLAM facilitated the suppression. These results suggest that MV, by altering DC function, renders them unresponsive to secondary pathogens via TLR4. [Copyright &y& Elsevier]

Abstract: Measles virus V protein is a Cys-rich polypeptide that is dispensable for virus propagation in continuous cell lines, but necessary for efficient viral replication in animals. Those functions modulating virus propagation in vivo are not understood completely, although V protein is known to interfere with the host interferon response and control of viral gene expression. The ability to modulate gene expression was investigated further with a minireplicon transient expression system in which V protein was found to repress reporter activity. Two regions of the polypeptide contributed to this repressive effect including the carboxy-terminus and a region conserved in morbillivirus V proteins located between amino acids 110–131, whereas domains known to mediate the interaction between V and the nucleocapsid (N) protein were not essential. Accumulation of encapsidated minigenome in transfected cells was inhibited by V protein suggesting that it acted as a repressor of genome replication thereby limiting availability of template for reporter gene mRNA transcription. [Copyright &y& Elsevier]

Abstract: Measles virus V protein represses genome replication through a poorly understood mechanism, which led us to investigate whether V protein might be an RNA-binding modulatory factor. Recombinant V protein, expressed from transfected HEp-2 cells or E. coli, formed protein–RNA complexes with poly-guanosine (poly-G) or poly-U linked to agarose beads. RNA binding was not exclusive to ribonucleotide homopolymers as complex formation between V protein and an RNA molecule equivalent to the 3′ terminal 107 bases of the measles virus genome was observed with an electrophoretic mobility shift assay (EMSA). The interaction with poly-G was used to further examine the RNA binding properties of V demonstrating that protein–RNA complex formation was dependent upon the unique Cys-rich carboxy terminus, a region also required to induce maximal repression of minireplicon-encoded reporter gene expression in transient assays. Surprisingly, two mutant proteins that contained Cys-to-Ala substitutions in the C-terminus were found to retain their ability to bind poly-G binding and repress minireplicon reporter gene expression indicating that neither activity was dependent on the integrity of all 7 C-terminal Cys residues. Additional genetic analysis revealed that amino acids 238–266 were necessary for efficient RNA binding and overlapped with residues (238–278) required for maximal repression induced by the C-terminal domain. In addition, a 10 amino acid deletion was identified (residues 238–247) that blocked RNA binding and repression indicating that these two activities were related. [Copyright &y& Elsevier]

Abstract: Measles virus (MV), one of the most infectious of human pathogens, still infects over 30 million humans and causes over 500,000 deaths each year [Griffin, D., 2001. Measles virus. In: Fields, B., Knipe, D., Howley, P. (Eds.), Fields Virology. Lippincott-Raven, Philadelphia, pp. 1401–1442; ]. Death is primarily due to secondary microbial infections associated with the immunosuppression caused by MV. Studies of humans with genetic or acquired deficiencies of either the humoral or cellular arm of the immune system, and rodent models have implicated T cells in the control of the ongoing MV infection but the precise role and activities of the specific T cell subset or the molecules they produce is not clear. Using a transgenic mouse model in conjunction with depletion and reconstitution of individual B and T cell subsets alone or in combination, we show that neither CD4, CD8 nor B cells per se control acute MV infection. However, combinations of either CD4 T cells and B cells, or of CD4 and CD8 T cells are essential but CD8 T with B cells are ineffective. Interferon-γ and neutralizing antibodies, but neither perforin nor TNF-α alone are associated with clearance of MV infection. TNF-α combined with interferon-γ is more effective in protection than interferon alone. Further, the lack of an interferon-γ response leads to persistence of MV. [Copyright &y& Elsevier]

Abstract: Measles virus belongs to the Paramyxoviridae family within the Mononegavirales order. Its non-segmented, single stranded, negative sense RNA genome is encapsidated by the nucleoprotein (N) to form a helical nucleocapsid. This ribonucleoproteic complex is the substrate for both transcription and replication. The RNA-dependent RNA polymerase binds to the nucleocapsid template via its co-factor, the phosphoprotein (P). In this review, we summarize the main experimental data pointing out the abundance of structural disorder within measles virus N and P. We also describe studies indicating that structural disorder is a widespread property in the replicative complex of Paramyxoviridae and, more generally, of Mononegavirales. The functional implications of structural disorder are also discussed. Finally, we propose a model where the flexibility of the disordered N and P domains allows the formation of a tripartite complex (N°–P–L) during replication, followed by the delivery of N monomers to the newly synthesized genomic RNA chain. [Copyright &y& Elsevier]

Abstract: The major inducible 70-kDa heat shock protein (hsp72) binds measles virus (MV) nucleocapsids and increases MV gene expression. The cytoplasmic tail of the MV N protein (NTAIL) contains three hydrophobic domains (Box-1–3) that are potential targets of hsp72 interaction. Low affinity binding to Box-3 is correlated to hsp72-dependent stimulation of MV minireplicon reporter gene expression whereas interactions between hsp72 and Box-1 and/or -2 have not been documented. The present work showed that virus deficient in Box-3/hsp72 interaction retains the ability to form nucleocapsid/hsp72 complexes, identifying Box-2 but not Box-1 as a mediator of high affinity hsp72 binding. Box-2 is the binding site for the viral P protein X domain (XD), where P tethers the viral polymerase to nucleocapsid in support of transcription and genome replication, and competitive inhibition of XD binding to NTAIL by hsp72 was shown. Recognition of a common binding site by P and hsp72 represents a potential mechanism for host cell modulation of viral gene expression. [Copyright &y& Elsevier]

Abstract: The C protein of measles virus (MV C) is a basic protein of 186 amino acids (aa) that plays at least two roles in infected cells, interference with the innate immune response and modulation of viral polymerase activity. In this study, Northern blots were used to demonstrate that C proteins from three vaccine strains and three wild-type isolates of MV downregulated both mRNA transcription and genome replication in a plasmid-based mini-genome assay. The effect on transcription always paralleled the effect on replication; however, the six MV C proteins varied considerably in their ability to inhibit polymerase activity. Though the amino-terminal 45 aa of the C protein are more variable among different MV strains than the remaining 75% of the protein, the ability of the MV C proteins to inhibit polymerase activity was not regulated by substitutions in the amino terminus, but rather by the more conserved region containing aa 46–167. Naturally occurring substitutions at positions 147 and 166, but not 88 and 186, were found to regulate MV C protein activity. Deletion of the carboxyl-terminal 19 aa did not affect the polymerase-modulating activity. Though we did not find a link between the aa changes in MV C and attenuation, these data provide new information regarding the functions of this non-structural protein. [Copyright &y& Elsevier]

Receptor specificity is a critical determinant of viral tropism, but the capacity of viruses to switch to alternative receptors has not been extensively studied. Here, we engineered the attachment protein of an attenuated measles virus and generated truly retargeted viruses that are blind to the native receptors CD46 and SLAM, but which propagate efficiently and exclusively via alternative cellular receptors, epidermal growth factor receptor, or CD38. The engineered receptor tropisms were stably maintained during multiple serial virus passage without reversion to native receptor usage, even on cells offering the choice of both native and alternative receptors. We conclude that paramyxoviruses have a remarkably flexible and adaptable entry mechanism. [Copyright &y& Elsevier]

Measles virus encodes an RNA-dependent RNA polymerase composed of the L and P proteins. Recent studies have shown that the L proteins of both Sendai virus and parainfluenza virus 3 form an L–L complex [Çevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very Nterminus of the Sendai virus L RNA polymerase protein. Virology 313, 525–536.; Smallwood, S., Moyer, S.A., 2004. The L polymerase protein of parainfluenza virus 3 forms anoligomer and can interact with the heterologous Sendai virus L, P and C proteins. Virology 318, 439–450.; Smallwood, S., Cevik, B., Moyer, S.A., 2002. Intragenic complementation and oligomerization of the L subunit of the Sendai virus RNA polymerase. Virology 304, 235–245.]. Using differentially tagged L proteins, we show here that measles L also forms an oligomer and the L–L binding site resides in the N-terminal 408 amino acids overlapping the P binding site in the same region of L. To identify amino acids important for binding P and L, site-directed mutagenesis of the L-408 protein was performed. Seven of twelve mutants in L-408 were unable to form a complex with measles P while the remainder did bind at least some P. In contrast, all of the mutants retained the ability to form the L–L complex, so different amino acids are involved in the L and P binding sites on L. Four of the 408 mutations defective in P binding were inserted into the full-length measles L protein and all retained L–L complex formation, but did not bind P. Full-length L mutants that did not bind P were also inactive in viral RNA synthesis, showing a direct correlation between P–L complex formation and activity. [Copyright &y& Elsevier]

Measles virus (MV) infects dendritic cells (DCs) resulting in immunosuppression. Human DCs express two MV receptors: CD46 and human signaling lymphocyte activation molecule (hSLAM); thus, the role played by either alone is unclear. Because wild-type (wt) MV uses hSLAM receptor preferentially, we dissected the molecular basis of MV–DC interaction and resultant immunosuppression through the hSLAM receptor by creating transgenic (tg) mice expressing hSLAM on DCs. After infection with wt MV, murine splenic DCs expressing hSLAM receptor had less B7-1, B7-2, CD40, MHC class I, and MHC class II molecules on their surfaces and displayed an increased rate of apoptosis when compared to uninfected DCs. Further, MV-infected DCs failed to stimulate allogeneic T cells and inhibited mitogen-dependent T-cell proliferation. Individual expression of human SLAM, interferon α/β receptor, tumor necrosis factor-α, and lymphotoxin-α or β from T cells was not required for MV-infected DCs to inhibit the proliferation of T cells. [Copyright &y& Elsevier]

Nuclear matrix is a structural framework of important nuclear processes. We studied the effect of two different types of viral infections on nuclear matrix. HeLa cells were infected with human rhinovirus 1B (HRV 1B) or measles virus (MV), and Nuclear Mitotic Apparatus protein (NuMA) and lamins A/C and B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. We show that NuMA, lamins, and poly(ADP-ribose) polymerase-1 are cleaved during viral infection in a virus family-specific manner suggesting that these viruses activate different sets of proteases. Morphologically, NuMA was excluded from the condensed chromatin, lamins showed a folded distribution, and both proteins finally remained around the nuclear fragments. A general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) prevented the nuclear disintegration and the cleavage of the proteins studied. Interestingly, z-VAD-FMK rescued MV-infected but not HRV 1B-infected cells from cell death. These results show for the first time that NuMA and lamins are specific target proteins during virus-induced programmed cell death. [Copyright &y& Elsevier]

Type I interferons (IFNα/β) are an important part of innate immunity to viral infections because they induce an antiviral response and limit viral replication until the adaptive response clears the infection. Since the nonstructural proteins of several paramyxoviruses inhibit the IFNα/β response, we chose to explore the role of the C protein of measles virus (MV) in such inhibition. Previous studies have suggested that the MV C protein may serve as a virulence factor, but its role in the pathogenesis of MV remains undefined. In the present study, a recombinant MV strain that does not express the C protein (MV C−) and its parental strain (Ed Tag) were used. Growth of MV C− was restricted in human peripheral blood mononuclear cells and HeLa cells, but in the presence of neutralizing antibodies to IFNα/β, MV C− produced titers that were equivalent to those of Ed Tag. In addition, expression of the MV C protein from plasmid DNA inhibited the production of an IFNα/β responsive reporter gene and, to a lesser extent, inhibited an IFNγ responsive reporter gene. The ability of the MV C protein to suppress the IFNα/β response was confirmed using a biologic assay. After IFNβ stimulation, HeLa cells infected with Ed Tag produced five-fold less IFNα/β than cells infected with MV C−. While the mechanism of inhibition remains unclear, these data suggest that the MV C protein plays an important role in the pathogenesis of MV by inhibiting IFNα/β signaling. [Copyright &y& Elsevier]

This is the first report of the direct identification of a HLA-DRB1*03 measles-derived peptide from measles virus infected EBV-transformed B cells. We purified HLA-DR3-peptide complexes from EBV-B cells infected with measles virus (Edmonston strain) and sequenced the HLA-DR3-peptides by mass spectrometry. A class II peptide, derived from a measles phosphoprotein, ASDVETAEGGEIHELLRLQ (P1, residues 179–197), exhibited the capacity to stimulate peripheral blood mononuclear cells to proliferate. Our data provides direct evidence that the antigenic peptide of measles virus was processed by antigen-presenting cells, presented in the context of HLA class II molecules, and was recognized by peripheral blood T cells from healthy individuals previously immunized with measles vaccine. The approach described herein provides a useful methodology for the future identification of HLA-presented pathogen-derived epitopes using mass spectrometry. The study of cell-mediated immune responses to the measles-derived peptide in immune persons should provide significant insight into the design and development of new vaccines. [Copyright &y& Elsevier]

Two nonrelated viruses, cowpea mosaic virus (wtCPMV) and measles virus (MV), were found to induce cross-reactive antibodies. The nature of this cross-reactivity was studied and results are presented here demonstrating that antiserum raised against wtCPMV reacted with peptide from the fusion (F) protein of MV. Furthermore, the F protein of MV was shown to share an identical conformational B cell epitope with the small subunit of CPMV coat protein. Passive transfer of anti-wtCPMV antibodies into BALB/c mice conferred partial protection against measles virus induced encephalitis. The results are discussed in the context of cross-protection. [Copyright &y& Elsevier]

To eradicate measles in developing nations a vaccine capable of being administered at birth may be necessary. We immunized newborn rhesus macaques with naked DNA encoding the measles virus hemagglutinin, fusion and nucleoprotein genes. Prior to vaccination we passively transferred measles immunoglobulin to mimic maternal antibody. In the presence or absence of measles immunoglobulin, 23 of 25 infant macaques had detectable cell mediated immunity and 16 had protective levels of neutralizing antibody. The co-administration of an IL-2/IgG plasmid augmented the vaccine, increasing cell mediated immunity in all infants and increasing the antibody response in infants vaccinated without immunoglobulin. We show for the first time that DNA vaccination can protect a newborn primate from the high-level viremia that correlates with severe measles, even in the presence of maternal antibody. Further, the addition of a molecular IL-2 adjuvant augments this DNA vaccine. [Copyright &y& Elsevier]

To establish infections, viruses use various strategies to suppress the host defense mechanism, such as interferon (IFN)-induced antiviral state. We found that cells infected with a wild strain of measles virus (MeV) displayed nearly complete suppression of IFN-α-induced antiviral state, but not IFN-γ-induced state. This phenomenon is due to the suppression of IFN-α-inducible gene expression at a transcriptional level. In the IFN-α signal transduction pathway, Jak1 phosphorylation induced by IFN-α is dramatically suppressed in MeV-infected cells; however, phosphorylation induced by IFN-γ is not. We performed immunoprecipitation experiments using antibodies against type 1 IFN receptor chain 1 (INFAR1) and antibody against RACK1, which is reported to be a scaffold protein interacting with type I IFN receptor chain 2 and STAT1. These experiments indicated that IFNAR1 forms a complex containing the MeV-accessory proteins C and V, RACK1, and STAT1 in MeV-infected cells but not in uninfected cells. Composition of this complex in the infected cells altered little by IFN-α treatment. These results indicate that MeV suppresses the IFN-α, but not IFN-γ, signaling pathway by inhibition of Jak1 phosphorylation. Our data suggest that functional disorder of the type I IFN receptor complex is due to “freezing” of the receptor through its association with the C and/or V proteins of MeV. [Copyright &y& Elsevier]

The nucleoprotein (N) of measles virus encapsidates viral genomic RNA to form a helical nucleocapsid. Its strong self-association is a major hurdle in determining its high-resolution structure using X-ray crystallography. We report the bacterial expression, purification, and characterization of a variant N that has lost its ability to form nucleocapsid-like structures after substitution of two residues by polar residues. Using immunoprecipitation, circular dichroism, and limited proteolysis studies, we show that this nucleoprotein retains a folding similar to wild-type N. Furthermore, the variant N binds the phosphoprotein, indicating that it retains biochemical relevance. We also present evidence indicating that the N-terminus of N lies at the surface of the nucleocapsid. Beyond the identification of one region of N involved in self-association, our results should facilitate structural studies of N using X-ray crystallography. [Copyright &y& Elsevier]