Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) encodes two transmembrane glycoproteins E1 and E2 which form a heterodimer. E1 is believed to mediate fusion while E2 has been shown to bind cellular receptors including CD81. In this study, alanine substitutions in E2 were generated within putative CD81 binding regions to define residues critical for viral entry. The effect of each mutation was tested by challenging susceptible cell lines with mutant HCV E1E2 pseudotyped viruses generated using a lentiviral system (HCVpp). In addition to assaying infectivity, producer cell expression and HCVpp incorporation of HCV E1 and E2 proteins, CD81 binding profiles, and E1E2 association of mutants were examined.</p> <p>Results</p> <p>Based on these characteristics, mutants either displayed wt characteristics (high infectivity [≥ 50% of wt HCVpp], CD81 binding, E1E2 expression, association, and incorporation into viral particles and proper conformation) or segregated into 4 distinct low infectivity (≤ 50% of wt HCVpp) mutant phenotypes: (I) CD81 binding deficient (despite wt E1E2 expression, incorporation and association and proper conformation); (II) CD81 binding competent, but lack of E1 detection on the viral particle, (despite adequate E1E2 expression in producer cell lysates and proper conformation); (III) CD81 binding competent, with adequate E1E2 expression, incorporation, association, and proper E2 conformation (i.e. no defect identified to explain the reduced infectivity observed); (IV) CD81 binding deficient due to disruption of E2 mutant protein conformation.</p> <p>Conclusion</p> <p>Although most alanine substitutions within the putative CD81 binding region 1 (amino acids 474–492) displayed greatly reduced HCVpp infectivity, they retained soluble CD81 binding, proper E2 conformation, E1E2 association and incorporation into HCVpp suggesting that region 1 of E2 does not mediate binding to CD81. In contrast, conformationally correct E2 mutants (Y527 and W529) within the second putative CD81 binding region (amino acids 522–551) disrupted binding of E2 to CD81-GST, suggesting that region 2 is critical to CD81 binding. Likewise, all conformationally intact mutants within the third putative CD81 binding region (amino acids 612–619), except L615A, were important for E2 binding to CD81-GST. This region is highly conserved across genotypes, underlining its importance in mediating viral entry.</p

Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20(th) century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism

Mutations in the NS1 C-terminal tail do not enhance replication or virulence of the 2009 pandemic H1N1 influenza A virus

The ‘classical’ swine H1N1 influenza A virus lineage was established after the devastating 1918 human pandemic virus entered domestic pig herds. A descendent of this lineage recently re-emerged in humans as the 2009 pandemic H1N1 virus. Adaptation in pigs has led to several changes in the multifunctional viral NS1 protein as compared with the parental 1918 virus, most notably a K217E substitution that abolishes binding to host Crk/CrkL signalling adapters, and an 11 aa C-terminal truncation. Using reverse genetics, we reintroduced both these features into a prototype 2009 H1N1 strain, A/California/04/09. Restoration of Crk/CrkL binding or extension of NS1 to 230 aa had no impact on virus replication in human or swine cells. In addition, minimal effects on replication, pathogenicity and transmission were observed in mouse and ferret models. Our data suggest that the currently circulating 2009 H1N1 virus is optimized to replicate efficiently without requiring certain NS1 functions

Cross-protection against European swine influenza viruses in the context of infection immunity against the 2009 pandemic H1N1 virus : studies in the pig model of influenza

Pigs are natural hosts for the same influenza virus subtypes as humans and are a valuable model for cross-protection studies with influenza. In this study, we have used the pig model to examine the extent of virological protection between a) the 2009 pandemic H1N1 (pH1N1) virus and three different European H1 swine influenza virus (SIV) lineages, and b) these H1 viruses and a European H3N2 SIV. Pigs were inoculated intranasally with representative strains of each virus lineage with 6- and 17-week intervals between H1 inoculations and between H1 and H3 inoculations, respectively. Virus titers in nasal swabs and/or tissues of the respiratory tract were determined after each inoculation. There was substantial though differing cross-protection between pH1N1 and other H1 viruses, which was directly correlated with the relatedness in the viral hemagglutinin (HA) and neuraminidase (NA) proteins. Cross-protection against H3N2 was almost complete in pigs with immunity against H1N2, but was weak in H1N1/pH1N1-immune pigs. In conclusion, infection with a live, wild type influenza virus may offer substantial cross-lineage protection against viruses of the same HA and/or NA subtype. True heterosubtypic protection, in contrast, appears to be minimal in natural influenza virus hosts. We discuss our findings in the light of the zoonotic and pandemic risks of SIVs

Immune Protection of Nonhuman Primates against Ebola Virus with Single Low-Dose Adenovirus Vectors Encoding Modified GPs

BACKGROUND: Ebola virus causes a hemorrhagic fever syndrome that is associated with high mortality in humans. In the absence of effective therapies for Ebola virus infection, the development of a vaccine becomes an important strategy to contain outbreaks. Immunization with DNA and/or replication-defective adenoviral vectors (rAd) encoding the Ebola glycoprotein (GP) and nucleoprotein (NP) has been previously shown to confer specific protective immunity in nonhuman primates. GP can exert cytopathic effects on transfected cells in vitro, and multiple GP forms have been identified in nature, raising the question of which would be optimal for a human vaccine. METHODS AND FINDINGS: To address this question, we have explored the efficacy of mutant GPs from multiple Ebola virus strains with reduced in vitro cytopathicity and analyzed their protective effects in the primate challenge model, with or without NP. Deletion of the GP transmembrane domain eliminated in vitro cytopathicity but reduced its protective efficacy by at least one order of magnitude. In contrast, a point mutation was identified that abolished this cytopathicity but retained immunogenicity and conferred immune protection in the absence of NP. The minimal effective rAd dose was established at 10(10) particles, two logs lower than that used previously. CONCLUSIONS: Expression of specific GPs alone vectored by rAd are sufficient to confer protection against lethal challenge in a relevant nonhuman primate model. Elimination of NP from the vaccine and dose reductions to 10(10) rAd particles do not diminish protection and simplify the vaccine, providing the basis for selection of a human vaccine candidate

Green Fluorescent Protein (GFP) Color Reporter Gene Visualizes Parvovirus B19 Non-Structural Segment 1 (NS1) Transfected Endothelial Modification

Background: Human Parvovirus B19 (PVB19) has been associated with myocarditis putative due to endothelial infection. Whether PVB19 infects endothelial cells and causes a modification of endothelial function and inflammation and, thus, disturbance of microcirculation has not been elucidated and could not be visualized so far. Methods and Findings: To examine the PVB19-induced endothelial modification, we used green fluorescent protein (GFP) color reporter gene in the non-structural segment 1 (NS1) of PVB19. NS1-GFP-PVB19 or GFP plasmid as control were transfected in an endothelial-like cell line (ECV304). The endothelial surface expression of intercellular-adhesion molecule-1 (CD54/ICAM-1) and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) were evaluated by flow cytometry after NS-1-GFP or control-GFP transfection. To evaluate platelet adhesion on NS-1 transfected ECs, we performed a dynamic adhesion assay (flow chamber). NS-1 transfection causes endothelial activation and enhanced expression of ICAM-1 (CD54: mean±standard deviation: NS1-GFP vs. control-GFP: 85.3±11.2 vs. 61.6±8.1; P<0.05) and induces endothelial expression of EMMPRIN/CD147 (CD147: mean±SEM: NS1-GFP vs. control-GFP: 114±15.3 vs. 80±0.91; P<0.05) compared to control-GFP transfected cells. Dynamic adhesion assays showed that adhesion of platelets is significantly enhanced on NS1 transfected ECs when compared to control-GFP (P<0.05). The transfection of ECs was verified simultaneously through flow cytometry, immunofluorescence microscopy and polymerase chain reaction (PCR) analysis. Conclusions: GFP color reporter gene shows transfection of ECs and may help to visualize NS1-PVB19 induced endothelial activation and platelet adhesion as well as an enhanced monocyte adhesion directly, providing in vitro evidence of possible microcirculatory dysfunction in PVB19-induced myocarditis and, thus, myocardial tissue damage

Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response.National Institutes of Health (U.S.

Assessing the Role of CD103 in Immunity to an Intestinal Helminth Parasite

In the intestine, the integrin CD103 is expressed on a subset of T regulatory (T(reg)) cells and a population of dendritic cells (DCs) that produce retinoic acid and promote immune homeostasis. However, the role of CD103 during intestinal helminth infection has not been tested.We demonstrate that CD103 is dispensable for the development of protective immunity to the helminth parasite Trichuris muris. While we observed an increase in the frequency of CD103(+) DCs in the lamina propria (LP) following acute high-dose infection with Trichuris, lack of CD103 had no effect on the frequency of CD11c(+) DCs in the LP or mesenteric lymph nodes (mLN). CD103-deficient (CD103(-/-)) mice develop a slightly increased and earlier T cell response but resolve infection with similar kinetics to control mice. Similarly, low-dose chronic infection of CD103(-/-) mice with Trichuris resulted in no significant difference in immunity or parasite burden. Absence of CD103 also had no effect on the frequency of CD4(+)CD25(+)Foxp3(+) T(reg) cells in the mLN or LP.These results suggest that CD103 is dispensable for intestinal immunity during helminth infection. Furthermore, lack of CD103 had no effect on DC or T(reg) recruitment or retention within the large intestine