Identification of Regulatory Host Genes Involved in Sigma Virus Replication Using RNAi Knockdown in Drosophila.

The Drosophila melanogaster sigma virus, a member of the Rhabdoviridae family, specifically propagates itself in D. melanogaster. It contains six genes in the order of 3'-N-P-X-M-G-L-5'. The sigma virus is the only arthropod-specific virus of the Rhabdoviridae family. Sigma-virus-infected Drosophila may suffer from irreversible paralysis when exposed to a high CO2 concentration, but generally, no other symptoms are reported. A recent study reported that host gene expression in immune pathways was not changed in sigma-virus-infected Drosophila, which does not necessarily suggest that they are not involved in virus-host interactions. The present study aimed to identify host genes associated with sigma virus replication. Immune pathways JAK-STAT and IMD were selected for detailed study. The results showed that the genome copy number of the sigma virus increased after knocking down the immune pathway genes domeless and PGRP-LC in Drosophila S2 cells. The knocking down of domeless and PGRP-LC significantly up-regulated the expression of the L gene compared to the other viral genes. We propose that the immune pathways respond to sigma virus infection by altering L expression, hence suppressing viral replication. This effect was further tested in vivo, when D. melanogaster individuals injected with dsdome and dsPGRP-LC showed not only an increase in sigma virus copy number, but also a reduced survival rate when treated with CO2. Our study proved that host immunity influences viral replication, even in persistent infection. Knocking down the key components of the immune process deactivates immune controls, thus facilitating viral expression and replication. We propose that the immunity system of D. melanogaster regulates the replication of the sigma virus by affecting the L gene expression. Studies have shown minimal host-virus interaction in persistent infection. However, our study demonstrated that the immunity continued to affect viral replication even in persistent infection because knocking down the key components of the immune process disabled the relevant immune controls and facilitated viral expression and replication.

Crystal structure of the C-terminal domain of the primosomal DnaT protein: Insights into a new oligomerization mechanism.

DnaT is a replication restart primosomal protein required for re-initiating chromosomal DNA replication in bacteria. DnaT can be a monomer, dimer, trimer, tetramer, or pentamer. The oligomerization and disassembly of DnaT oligomers are critical in primosome assembly. Prior to this work, only the ssDNA-bound structure of the pentameric DnaT truncated protein (aa 84-153; DnaT84-153) was available. The mechanism by which DnaT oligomerizes as different states is unclear. In this paper, we report the crystal structure of the C-terminal domain of DnaT (aa 84-179; DnaTc) at 2.30 Šresolution (PDB entry 6AEQ). DnaTc forms a dimer both in the crystalline state and in solution. As compared with the ssDNA-bound structure of the pentameric DnaT84-153, their subunit-subunit interfaces significantly differ. The different oligomeric architecture suggests a strong conformational change possibly induced by ssDNA. Superposition analysis further indicated that the monomer of a DnaTc dimer shifted away by a distance of 7.5 Šand rotated by an angle of 170° for binding to ssDNA. Basing from these molecular evidence, we discussed and proposed a working model to explain how DnaTc oligomerizes through residue R146 mediation.