West Nile Virus Infection in Plasma of Blood and Plasma Donors, United States

This study investigated the association of ongoing West Nile virus (WNV) infections with neutralizing antibody titers in US plasma-derived intravenous immune globulin released during 2003–2008. Titers correlated closely with the prevalence of past WNV infection in blood donors, with 2008 lots indicating a prevalence of 1%

Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly

Ebola Virus Transcription Activator VP30 Is a Zinc-Binding Protein

Ebola virus VP30 is an essential activator of viral transcription. In viral particles, VP30 is closely associated with the nucleocapsid complex. A conspicuous structural feature of VP30 is an unconventional zinc-binding Cys(3)-His motif comprising amino acids 68 to 95. By using a colorimetric zinc-binding assay we found that the VP30-specific Cys(3)-His motif stoichiometrically binds zinc ions in a one-to-one relationship. Substitution of the conserved cysteines and the histidine within the motif led to a complete loss of the capacity for zinc binding. Functional analyses revealed that none of the tested mutations of the proposed zinc-coordinating residues influenced binding of VP30 to nucleocapsid-like particles but, concerning its role in activating viral transcription, all resulted in a protein that was inactive

Ebola Virus VP30-Mediated Transcription Is Regulated by RNA Secondary Structure Formation

The nucleocapsid protein VP30 of Ebola virus (EBOV), a member of the Filovirus family, is known to act as a transcription activator. By using a reconstituted minigenome system, the role of VP30 during transcription was investigated. We could show that VP30-mediated transcription activation is dependent on formation of a stem-loop structure at the first gene start site. Destruction of this secondary structure led to VP30-independent transcription. Analysis of the transcription products of bicistronic minigenomes with and without the ability to form the secondary structure at the first transcription start signal revealed that transcription initiation at the first gene start site is a prerequisite for transcription of the second gene, independent of the presence of VP30. When the transcription start signal of the second gene was exchanged with the transcription start signal of the first gene, transcription of the second gene also was regulated by VP30, indicating that the stem-loop structure of the first transcription start site acts autonomously and independently of its localization on the RNA genome. Our results suggest that VP30 regulates a very early step of EBOV transcription, most likely by inhibiting pausing of the transcription complex at the RNA structure of the first transcription start site