C. Cheng Kao

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.

RNA viruses employ a combination of mechanisms to regulate their gene expression and replication. Brome mosaic virus (BMV) is a tripartite positive-strand RNA virus used to study the requirements for virus infection. BMV genomic RNA1 encodes protein 1a, which contains a methyltransferase (MT) domain and a helicase domain that are required for replication. 1a forms a complex with the 2a RNA-dependent RNA polymerase for the replication and transcription of all BMV RNAs. RNA1 expressed with 2a from Agrobacterium -based vectors can result in RNA1 replication in Nicotiana benthamiana. A mutation in the 1a translation initiation codon significantly decreased RNA1 accumulation even when wild-type (WT) 1a and 2a were provided in trans . Therefore, efficient RNA1 replication requires 1a translation from RNA1 in cis , indicating a linkage between replication and translation. Mutation analyses showed that the full-length 1a protein was required for efficient RNA1 replication, not just the process of translation. Three RNA1s with mutations in the 1a MT domain could be partially rescued by WT 1a expressed in trans , indicating that the cis -acting function of 1a was retained. Furthermore, an RNA motif in the 5′-untranslated region of RNA1, named the B box, was required for 1a to function in cis and in trans for BMV RNA accumulation. The B box is required for the formation of the replication factory (M. Schwartz, J. Chen, M. Janda, M. Sullivan, J. den Boon, and P. Ahlquist, Mol. Cell 9:505-514, 2002). Results in this work demonstrate a linkage between BMV RNA1 translation and replication.

The goal of this project was to better define the relationship between the endoribonuclease activity of murine hepatitis virus (MHV) Nsp15 (mNsp15) and its role in virus infection. Molecular modeling demonstrated that the catalytic residues of mNsp15 are superimposable with its severe acute respiratory syndrome coronavirus ortholog. Alanine substitutions at three key residues in the mNsp15 catalytic pocket (H262, H277, and G275) and a double-mutant version (H262P and H277A) generated proteins with greatly reduced but detectable endoribonuclease activities. Furthermore, these mutant proteins demonstrated lower cleavage specificities for uridylate than wild-type (WT) mNsp15. These mutations were successfully incorporated into viruses named vH262A, vH277A, vG275A, and vH262P+H277A. All four mutant viruses formed plaques with diameters similar to that of MHV-A59 1000 (WT virus) on several different cell lines. Interestingly, viruses with a mutation at a noncatalytic residue, D324A, could not be recovered despite repeated attempts, and expression of mNsp15 containing the D324A mutation in Escherichia coli resulted in an insoluble protein. Plaques derived from vH262A produced approximately 6- to 13-fold fewer PFU than those from WT virus. Cells infected with mNsp15 mutant viruses accumulated lesser amounts of plus- and minus-sense subgenomic RNAs and spike protein than WT virus. The expression of mNsp15 in trans by transient transfection partially restored RNA synthesis by vH262A. These results demonstrate that mNsp15 is required for optimal infection by MHV.

Viruses with separately encapsidated genomes could have their genomes introduced into different leaves of a plant, thus necessitating long-distance trafficking of the viral RNAs for successful infection. To examine this possibility, individual or combinations of genome segments from the tripartite Brome mosaic virus (BMV) were transiently expressed in leaves of Nicotiana benthamiana plants using engineered Agrobacterium tumefaciens. BMV RNA3 was found to traffic from the initial site of expression to other leaves of the plant, as detected by RNA gel blot analyses and also by the expression of an endoplasmic reticulum–targeted green fluorescent protein. When RNA3 trafficked into leaves containing the BMV replication enzymes, RNA replication, transcription, and virion production were observed. RNA3 trafficking occurred even when it did not encode the movement or capsid proteins. However, coexpression of the movement protein increased the trafficking of BMV RNAs. BMV RNA1 and RNA2 could also traffic throughout the plant, but less efficiently than RNA3. All three BMV RNAs trafficked bidirectionally to sink leaves near the apical meristem as well as to the source leaves at the bottom of the stem, suggesting that trafficking used the phloem. These results demonstrate that BMV RNAs can use a replication-independent mechanism to traffic in N. benthamiana.