A "coiled-coil" motif is important for oligomerization and DNA binding properties of human cytomegalovirus protein UL77.

Human cytomegalovirus (HCMV) UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM) at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM) implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE) was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0 ± 0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2 ± 0.41 and 4.9 ± 0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i) could form homodimers, (ii) CCM of pUL77 is crucial for oligomerization and (iii) could bind to dsDNA in a sequence independent manner.

Analysis of different DNA vaccines for protection of experimental influenza A virus infection.

Influenza viruses cause acute respiratory infections in humans that result in significant excessive morbidity and mortality rates every year. Current vaccines are limited in several aspects, including laborious manufacturing technology, non-sufficient efficacy, and time-consuming adjustments to new emerging virus variants. An alternative vaccine approach utilizes plasmid DNA encoding influenza virus antigens. Previous experiments have evaluated the protective efficacy of DNA vaccines expressing variable as well as conserved antigens. In this present study, several different combinations of influenza A virus (IAV) HA, NA, M1, M2, NS1, NS2, and NP sequences were cloned into the plasmid pVIVO, which allows the independent expression of two genes separately. These DNA vaccines were administered to induce protection against a lethal IAV infection, and to reduce immunopathology in lung tissue of surviving animals. The highest efficacy was provided by vaccines expressing HA and NA, as well as a mixture of plasmids encoding HA, NA, M1, M2, NS1, NS2, and NP (Mix). Three days post-infection, more than a 99.99% reduction of viral load and no inflammation was achieved in lung tissue of pVIVO/HA-NA-vaccinated mice. Animals vaccinated with pVIVO/HA-NA, pVIVO/HA-M2, or vaccine Mix, survived a lethal challenge with minor or no obvious pathologic abnormities in the lungs. All other surviving mice revealed extensive changes in the lung tissue, indicating possibly an ongoing bronchiolitis obliterans. In addition, pVIVO/HA-NA and the vaccine Mix were also protective against a heterologous IAV infection. Taken together, next to all combinations of different DNA vaccines, the intramuscular application of pVIVO/HA-NA was the most efficient procedure to decrease virus replication and to prevent immunopathology in lung tissue of IAV-infected mice.