Expression of Human CD4 and chemokine receptors in cotton rat cells confers permissiveness for productive HIV infection

<p>Abstract</p> <p>Background</p> <p>Current small animal models for studying HIV-1 infection are very limited, and this continues to be a major obstacle for studying HIV-1 infection and pathogenesis, as well as for the urgent development and evaluation of effective anti-HIV-1 therapies and vaccines. Previously, it was shown that HIV-1 can infect cotton rats as indicated by development of antibodies against all major proteins of the virus, the detection of viral cDNA in spleen and brain of challenged animals, the transmission of infectious virus, albeit with low efficiency, from animal to animal by blood, and an additional increase in the mortality in the infected groups.</p> <p>Results</p> <p>Using <it>in vitro </it>experiments, we now show that cotton rat cell lines engineered to express human receptor complexes for HIV-1 (hCD4 along with hCXCR4 or hCCR5) support virus entry, viral cDNA integration, and the production of infectious virus.</p> <p>Conclusion</p> <p>These results further suggest that the development of transgenic cotton rats expressing human HIV-1 receptors may prove to be useful small animal model for HIV infection.</p

A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

<p>Abstract</p> <p>Background</p> <p>Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new <it>Henipavirus </it>genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.</p> <p>Results</p> <p>Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.</p> <p>Conclusions</p> <p>Together, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.</p

Residues in the Stalk Domain of the Hendra Virus G Glycoprotein Modulate Conformational Changes Associated with Receptor Binding▿

Hendra virus (HeV) is a member of the broadly tropic and highly pathogenic paramyxovirus genus Henipavirus. HeV is enveloped and infects cells by using membrane-anchored attachment (G) and fusion (F) glycoproteins. G possesses an N-terminal cytoplasmic tail, an external membrane-proximal stalk domain, and a C-terminal globular head that binds the recently identified receptors ephrinB2 and ephrinB3. Receptor binding is presumed to induce conformational changes in G that subsequently trigger F-mediated fusion. The stalk domains of other attachment glycoproteins appear important for oligomerization and F interaction and specificity. However, this region of G has not been functionally characterized. Here we performed a mutagenesis analysis of the HeV G stalk, targeting a series of isoleucine residues within a hydrophobic α-helical domain that is well conserved across several attachment glycoproteins. Nine of 12 individual HeV G alanine substitution mutants possessed a complete defect in fusion-promotion activity yet were cell surface expressed and recognized by a panel of conformation-dependent monoclonal antibodies (MAbs) and maintained their oligomeric structure. Interestingly, these G mutations also resulted in the appearance of an additional electrophoretic species corresponding to a slightly altered glycosylated form. Analysis revealed that these G mutants appeared to adopt a receptor-bound conformation in the absence of receptor, as measured with a panel of MAbs that preferentially recognize G in a receptor-bound state. Further, this phenotype also correlated with an inability to associate with F and in triggering fusion even after receptor engagement. Together, these data suggest the stalk domain of G plays an important role in the conformational stability and receptor binding-triggered changes leading to productive fusion, such as the dissociation of G and F

New Insights into the Hendra Virus Attachment and Entry Process from Structures of the Virus G Glycoprotein and Its Complex with Ephrin-B2

<div><p>Hendra virus and Nipah virus, comprising the genus <em>Henipavirus</em>, are recently emerged, highly pathogenic and often lethal zoonotic agents against which there are no approved therapeutics. Two surface glycoproteins, the attachment (G) and fusion (F), mediate host cell entry. The crystal structures of the Hendra G glycoprotein alone and in complex with the ephrin-B2 receptor reveal that henipavirus uses Tryptophan 122 on ephrin-B2/B3 as a “latch” to facilitate the G-receptor association. Structural-based mutagenesis of residues in the Hendra G glycoprotein at the receptor binding interface document their importance for viral attachments and entry, and suggest that the stability of the Hendra-G-ephrin attachment complex does not strongly correlate with the efficiency of viral entry. In addition, our data indicates that conformational rearrangements of the G glycoprotein head domain upon receptor binding may be the trigger leading to the activation of the viral F fusion glycoprotein during virus infection.</p> </div

Four hydrophobic residues of the ephrin-B2 G-H loop insert in four hydrophobic HeV-G pockets.

<p>The four ephrin residues (F117, P119, L121 and W122) are illustrated as purple sticks. The HeV-G pockets are shown as a yellow surface. The residues defining these pockets are shown as yellow lines and are labeled.</p

Structure of the HeV-G dimer.

<p>The secondary structure elements of the two molecules are colored in cyan and green. The axes of the two six-blade β-propellers are approximately perpendicular to each other. Disulfide bonds are illustrated as yellow sticks. Asparagine-linked carbohydrate modifications (glycosylations) are illustrated as grey spheres.</p

Interaction of HeV-G mutants with HeV-F.

<p>(A) HeV-G mutants were co expressed with S tagged HeV-F in HeLa-USU cells. Lysates were immunoprecipitated (IP) with rabbit polyclonal G-specific antiserum to evaluate total G expression (top panel) or S agarose to evaluate total F expression (bottom panel) and co-precipitation of G (middle panel). The precipitated products were analyzed on SDS-PAGE under reducing conditions and then western blot analyzed with F (bottom panel) or G (top and middle panel) specific mouse mAbs. The western blot result of one of three independent experiments is shown in (B). The relative HeV-F binding ability of each HeV-G mutant is shown in comparison to that of WT HeV-G and normalized to total HeV-G expression and the means of three independent experiments are shown. Error bars represent the ranges. The results were calculated using values obtained from digital densitometric measurements of the images.</p

Structure of the HeV-G/ephrin-B2 complex.

<p>The HeV-G and ephrin-B2 molecule are colored in yellow and purple, respectively. Ephrin-B2 sits on top face of the HeV-G β-propeller. The G-H loop of ephrin-B2 extends into the central cavity of HeV-G's β-propeller barrel.</p

Comparison of the NiV-G/ephrin-B3 and HeV-G/ephrin-B2 interfaces.

<p>HeV-G residues are colored in yellow; NiV-G is in cyan. The changes V/T507, F/Y458, I/V401 alter the hydrophobicity of the interface and thus contribute to the different receptor-binding affinities of NiV-G and HeV-G.</p