Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

AbstractThe 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

Elements in the Canine Distemper Virus M 3′ UTR Contribute to Control of Replication Efficiency and Virulence

Canine distemper virus (CDV) is a negative-sense, single-stranded RNA virus within the genus Morbillivirus and the family Paramyxoviridae. The Morbillivirus genome is composed of six transcriptional units that are separated by untranslated regions (UTRs), which are relatively uniform in length, with the exception of the UTR between the matrix (M) and fusion (F) genes. This UTR is at least three times longer and in the case of CDV also highly variable. Exchange of the M-F region between different CDV strains did not affect virulence or disease phenotype, demonstrating that this region is functionally interchangeable. Viruses carrying the deletions in the M 3′ UTR replicated more efficiently, which correlated with a reduction of virulence, suggesting that overall length as well as specific sequence motifs distributed throughout the region contribute to virulence

A Novel Ribozyme-Based Prophylaxis Inhibits Influenza A Virus Replication and Protects from Severe Disease

Influenza A virus seasonal outbreaks and occasional pandemics represent a global health threat. The high genetic instability of this virus permits rapid escape from the host immune system and emergence of resistance to antivirals. There is thus an urgent need to develop novel approaches for efficient treatment of newly emerging strains. Based on a sequence alignment of representatives from every subtype known to infect humans, we identified nucleic acid regions that are conserved amongst these influenza A populations. We then engineered SOFA-HDV-Ribozymes as therapeutic tools recognizing these conserved regions to catalytically cleave the corresponding viral mRNA targets. The most promising ribozymes were chosen based on an initial in silico screening, and their efficacy was assessed using in vitro cleavage assays. Further characterization of their antiviral effect in cell culture and in mice led to the gradual identification of prophylactic SOFA-HDV-Ribozyme combinations, providing proof-of-principle for the potential of this novel strategy to develop antivirals against genetically highly variable viruses

Generation of therapeutic antisera for emerging viral infections

The recent Ebola virus outbreak has highlighted the therapeutic potential of antisera and renewed interest in this treatment approach. While human convalescent sera may not be readily available in the early stages of an outbreak, antisera of animal origin can be produced in a short time frame. Here, we compared adjuvanted virus-like particles (VLP) with recombinant modified vaccinia virus Ankara and vesicular stomatitis virus (VSV), both expressing the Ebola virus antigens. The neutralizing antibody titers of rabbits immunized with adjuvanted VLPs were similar to those immunized with the replication-competent VSV, indicating that presentation of the antigen in its native conformation rather than de novo antigen expression is essential for production of functional antibodies. This approach also yielded high-titer antisera against Nipah virus glycoproteins, illustrating that it is transferable to other virus families. Multiple-step immunoglobulin G purification using a two-step 20-40% ammonium sulfate precipitation followed by protein A affinity chromatography resulted in 90% recovery of functionality and sustained in vivo stability. Adjuvanted VLP-based immunization strategies are thus a promising approach for the rapid generation of therapeutic antisera against emerging infections

Adjuvant formulated virus-like particles expressing native-like forms of the Lassa virus envelope surface glycoprotein are immunogenic and induce antibodies with broadly neutralizing activity

Lassa mammarenavirus (LASV) is a rodent-borne arenavirus endemic to several West African countries. It is the causative agent of human Lassa fever, an acute viral hemorrhagic fever disease. To date, no therapeutics or vaccines against LASV have obtained regulatory approval. Polyclonal neutralizing antibodies derived from hyperimmunized animals may offer a useful strategy for prophylactic and therapeutic intervention to combat human LASV infections. The LASV envelope surface glycoprotein complex (GP) is the major target for neutralizing antibodies, and it is the main viral antigen used for the design of an LASV vaccine. Here, we assessed the immunogenic potential of mammalian cell-derived virus-like particles (VLPs) expressing GP from the prototypic LASV strain Josiah in a native-like conformation as the sole viral antigen. We demonstrate that an adjuvanted prime-boost immunization regimen with GP-derived VLPs elicited neutralizing antibody responses in rabbits, suggesting that effective antigenic epitopes of GP were displayed. Notably, these antibodies exhibited broad reactivity across five genetic lineages of LASV. VLP-based immunization strategies may represent a powerful approach for generating polyclonal sera containing cross-reactive neutralizing antibodies against LASV

Zika virus infection elicits auto-antibodies to C1q

Zika virus (ZIKV) causes mostly asymptomatic infection or mild febrile illness. However, with an increasing number of patients, various clinical features such as microcephaly, Guillain-Barré syndrome and thrombocytopenia have also been reported. To determine which host factors are related to pathogenesis, the E protein of ZIKV was analyzed with the Informational Spectrum Method, which identifies common information encoded by primary structures of the virus and the respective host protein. The data showed that the ZIKV E protein and the complement component C1q cross-spectra are characterized by a single dominant peak at the frequency F = 0.338, suggesting similar biological properties. Indeed, C1q-specific antibodies were detected in sera obtained from mice and monkeys infected with ZIKV. As C1q has been known to be involved not only in immunity, but also in synaptic organization and different autoimmune diseases, a ZIKV-induced anti-C1q antibody response may contribute to the neurological complications. These findings might also be exploited for the design of safe and efficacious vaccines in the future

Amino-Terminal Precursor Sequence Modulates Canine Distemper Virus Fusion Protein Function

The fusion (F) proteins of most paramyxoviruses are classical type I glycoproteins with a short hydrophobic leader sequence closely following the translation initiation codon. The predicted reading frame of the canine distemper virus (CDV) F protein is more complex, with a short hydrophobic sequence beginning 115 codons downstream of the first AUG. To verify if the sequence between the first AUG and the hydrophobic region is translated, we produced a specific antiserum that indeed detected a short-lived F protein precursor that we named PreF(0). A peptide resulting from PreF(0) cleavage was identified and named Pre, and its half-life was measured to be about 30 min. PreF(0) cleavage was completed before proteolytic activation of F(0) into its F(1) and F(2) subunits by furin. To test the hypothesis that the Pre peptide may influence protein activity, we compared the function of F proteins synthesized with that peptide to that of F proteins synthesized with a shorter amino-terminal signal sequence. F proteins synthesized with the Pre peptide were more stable and less active. Thus, the Pre peptide modulates the function of the CDV F protein. Interestingly, a distinct two-hit activation process has been recently described for human respiratory syncytial virus, another paramyxovirus

N-Linked Glycans with Similar Location in the Fusion Protein Head Modulate Paramyxovirus Fusion

N-linked glycans not only orchestrate the folding and intracellular transport of viral glycoproteins but also modulate their function. We have characterized the three glycans attached to fusion (F) proteins of the morbilliviruses canine distemper virus and measles virus. The individual Morbillivirus glycans have similar functional properties: the glycan at position 68 is essential for protein transport, and those at positions 36 and 75 modulate fusion (numbering according to the Newcastle disease virus [NDV] F protein sequence). Based on the crystal structure of the NDV F protein, we then predicted the locations of the Morbillivirus glycans: the glycan at position 36 is located in the F protein head, and those at positions 68 and 75 are located near the neck-stalk interface. NDV position 36 is not occupied by a glycan; the only glycan in that F protein head also has a fusion control function and grows from residue 366, located only 6 Å from residue 36. We then exchanged the glycan at position 36 with the glycan at position 366 and showed functional complementation. Thus, structural information about the F proteins of Paramyxoviridae coupled with functional analysis disclosed a location in the protein head into which fusion-modulating glycans independently evolved