A Sendai virus recombinant vaccine expressing a gene for truncated human metapneumovirus (hMPV) fusion protein protects cotton rats from hMPV challenge.

Human metapneumovirus (hMPV) infections pose a serious health risk to young children, particularly in cases of premature birth. No licensed vaccine exists and there is no standard treatment for hMPV infections apart from supportive hospital care. We describe the production of a Sendai virus (SeV) recombinant that carries a gene for a truncated hMPV fusion (F) protein (SeV-MPV-Ft). The vaccine induces binding and neutralizing antibody responses toward hMPV and protection against challenge with hMPV in a cotton rat system. Results encourage advanced development of SeV-MPV-Ft to prevent the morbidity and mortality caused by hMPV infections in young children.

Residues in the heptad repeat a region of the fusion protein modulate the virulence of Sendai virus in mice.

While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.

Peptide-conjugated quantum dots: imaging the angiotensin type 1 receptor in living cells.

Peptide-quantum dot conjugates have been prepared by attaching angiotensin II (Ang II) to cadmium selenide/zinc sulfide core-shell nanocrystals using an 1-[3-(Dimethyamino)propyl]-3-ethylcarbo diimide hydrochloride (EDC) coupling. These conjugates have been used to image angiotensin I-expressing Chinese hamster ovary (CHO) cells in vitro. When CHO cells were incubated with Ang II before incubating with Ang II-conjugated quantum dots, we were able to block the binding of the dots. The Ang II-quantum dot conjugates did not bind to parental cells and showed similar staining patterns when compared with a commercially available Ang II Alexa 488 conjugate.

Regulation of dopamine D(1) receptor trafficking by protein kinase A-dependent phosphorylation.

The aim of this study was to use pharmacological inhibition of protein kinase A and mutation of potential protein kinase A phosphorylation sites to determine the role of protein kinase A-catalyzed phosphorylation of the dopamine D(1) receptor in agonist-stimulated desensitization and internalization of the receptor. To facilitate purification and imaging of the D(1) receptor, we attached a polyhistidine tag to the amino terminus and enhanced green fluorescent protein to the carboxyl terminus of the receptor (D(1)-EGFP). D(1)-EGFP was similar to the untagged D(1) receptor in terms of affinity for agonist and antagonist ligands, coupling to G proteins, and stimulation of cyclic AMP accumulation. D(1)-EGFP and two mutants in which either Thr268 or Ser380 was replaced with Ala were stably expressed in NS20Y neuroblastoma cells. Pretreatment with the protein kinase A inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) or substitution of Ala for Thr268 reduced agonist-stimulated phosphorylation of the receptor and resulted in diminished trafficking of the receptor to the perinuclear region of the cell. Substitution of Ala for Thr268 had no effect, however, on agonist-induced receptor sequestration or desensitization of cyclic AMP accumulation. Substitution of Ala for Ser380 had no effect on D(1) receptor phosphorylation, sequestration, desensitization, or trafficking to the perinuclear region. We conclude that protein kinase A-dependent phosphorylation of the D(1) receptor on Thr268 regulates a late step in the sorting of the receptor to the perinuclear region of the cell, but that phosphorylation of Thr268 is not required for receptor sequestration or maximal desensitization of cyclic AMP accumulation.