Triad of human cellular proteins, IRF2, FAM111A, and RFC3, restrict replication of orthopoxvirus SPI-1 host-range mutants.

Viruses and their hosts can reach balanced states of evolution ensuring mutual survival, which makes it difficult to appreciate the underlying dynamics. To uncover hidden interactions, virus mutants that have lost defense genes may be used. Deletion of the gene that encodes serine protease inhibitor 1 (SPI-1) of rabbitpox virus and vaccinia virus, two closely related orthopoxviruses, prevents their efficient replication in human cells, whereas certain other mammalian cells remain fully permissive. Our high-throughput genome-wide siRNA screen identified host factors that prevent reproduction and spread of the mutant viruses in human cells. More than 20,000 genes were interrogated with individual siRNAs and those that prominently increased replication of the SPI-1 deletion mutant were subjected to a secondary screen. The top hits based on the combined data-replication factor C3 (RFC3), FAM111A, and interferon regulatory factor 2 (IRF2)-were confirmed by custom assays. The siRNAs to RFC1, RFC2, RFC4, and RFC5 mRNAs also enhanced spread of the mutant virus, strengthening the biological significance of the RFC complex as a host restriction factor for poxviruses. Whereas association with proliferating cell nuclear antigen and participation in processive genome replication are common features of FAM111A and RFC, IRF2 is a transcriptional regulator. Microarray analysis, quantitative RT-PCR, and immunoblotting revealed that IRF2 regulated the basal level expression of FAM111A, suggesting that the enhancing effect of depleting IRF2 on replication of the SPI-1 mutant was indirect. Thus, the viral SPI-1 protein and the host IRF2, FAM111A, and RFC complex likely form an interaction network that influences the ability of poxviruses to replicate in human cells.

Intranuclear targeting of DNA replication factors.

Mammalian nuclei are highly organized into functional compartments. Major nuclear processes like DNA replication and RNA processing take place in distinct foci. These microscopically visible foci are formed by the assembly of, for example, DNA replication factors and associated proteins into megadalton complexes often referred to as protein machines or factories. Thus far, two proteins, DNA ligase I and DNA methyltransferase (DNA MTase), have been analyzed in greater detail. In both cases, the assembly process appears to be controlled by distinct targeting sequences that were attached to the catalytic protein core in the course of evolution and mediate the association with replication factories in mammalian cells. The dynamics of these nuclear structures throughout the cell cycle are analyzed using green fluorescent protein (GFP). Further studies are needed to elucidate the architecture, regulation, and role of these subnuclear structures. J. Cell. Biochem. Suppls. 30/31:243-249, 1998. © 1998 Wiley-Liss, Inc.