Complete deletion of Ectromelia virus p28 impairs virus genome replication in a mouse strain, cell type, and multiplicity of infection-dependent manner.

p28 is a poxvirus-encoded E3 ubiquitin ligase that possesses an N-terminal KilA-N domain and a C-terminal RING domain. In Ectromelia virus (ECTV), disruption of the p28 RING domain severely attenuated virulence in A strain mice, which normally succumb to ECTV infection. Moreover, this mutant virus exhibited dramatically reduced genome replication and impaired factory formation in A strain mice peritoneal macrophages (PMs) infected at high multiplicity of infection (MOI) These defects were not observed in PMs isolated from C57BL/6 mice which survive ECTV infection, demonstrating that p28 functions in a context-specific manner. To further investigate p28 function, we completely deleted the p28 gene from ECTV (ECTV-Δp28). In contrast to previous findings, we found that the ECTV-Δp28 virus exhibited severely compromised virus production and genome replication in PMs isolated from A strain mice only when infected at low MOI. This defect was minimal in bone marrow-derived macrophages and two cell lines derived from A strain mice. Furthermore, this low MOI defect in virus production was also observed in PMs isolated from the susceptible BALB/c mouse strain, but not PMs isolated from C57BL/6 mice. Taken together, our data demonstrate that the requirement for ECTV p28 to establish a productive infection depends on the MOI, the cell type, as well as the mouse strain.

Flavivirus Capsid Proteins Inhibit the Interferon Response.

Zika virus (ZIKV) establishes persistent infections in multiple human tissues, a phenomenon that likely plays a role in its ability to cause congenital birth defects and neurological disease. Multiple nonstructural proteins encoded by ZIKV, in particular NS5, are known to suppress the interferon (IFN) response by attacking different steps in this critical antiviral pathway. Less well known are the potential roles of structural proteins in affecting the host immune response during ZIKV infection. Capsid proteins of flaviviruses are of particular interest because a pool of these viral proteins is targeted to the nuclei during infection and, as such, they have the potential to affect host cell gene expression. In this study, RNA-seq analyses revealed that capsid proteins from six different flaviviruses suppress expression of type I IFN and IFN-stimulated genes. Subsequent interactome and in vitro ubiquitination assays showed that ZIKV capsid protein binds to and prevents activating ubiquitination of RIG-I CARD domains by TRIM25, a host factor that is important for the induction arm of the IFN response. The other flavivirus capsid proteins also interacted with TRIM25, suggesting that these viral proteins may attenuate antiviral signaling pathways at very early stages of infection, potentially even before nonstructural proteins are produced.

Vaccinia Virus Gene Acquisition through Nonhomologous Recombination.

Many of the genes encoded by poxviruses are orthologs of cellular genes. These virus genes serve different purposes, but perhaps of most interest is the way some have been repurposed to inhibit the antiviral pathways that their cellular homologs still regulate. What is unclear is how these virus genes were acquired, although it is presumed to have been catalyzed by some form(s) of nonhomologous recombination (NHR). We used transfection assays and substrates encoding a fluorescent and drug-selectable marker to examine the NHR frequency in vaccinia virus (VAC)-infected cells. These studies showed that when cells were transfected with linear duplex DNAs bearing VAC N2L gene homology, it yielded a recombinant frequency (RF) of 6.7 × 10-4. In contrast, DNA lacking any VAC homology reduced the yield of recombinants ∼400-fold (RF = 1.6 × 10-6). DNA-RNA hybrids were also substrates, although homologous molecules yielded fewer recombinants (RF = 2.1 × 10-5), and nonhomologous substrates yielded only rare recombinants (RF ≤ 3 × 10-8). NHR was associated with genome rearrangements ranging from simple insertions with flanking sequence duplications to large-scale indels that produced helper-dependent viruses. The insert was often also partially duplicated and would rapidly rearrange through homologous recombination. Most of the virus-insert junctions exhibited little or no preexiting microhomology, although a few encoded VAC topoisomerase recognition sites (C/T·CCTT). These studies show that VAC can catalyze NHR through a process that may reflect a form of aberrant replication fork repair. Although it is less efficient than classical homologous recombination, the rates of NHR may still be high enough to drive virus evolution. IMPORTANCE Large DNA viruses sometimes interfere in antiviral defenses using repurposed and mutant forms of the cellular proteins that mediate these same reactions. Such virus orthologs of cellular genes were presumably captured through nonhomologous recombination, perhaps in the distant past, but nothing is known about the processes that might promote "gene capture" or even how often these events occur over the course of an infectious cycle. This study shows that nonhomologous recombination in vaccinia virus-infected cells is frequent enough to seed a small but still significant portion of novel recombinants into large populations of newly replicated virus particles. This offers a route by which a pool of virus might survey the host genome for sequences that offer a selective growth advantage and potentially drive discontinuous virus evolution (saltation) through the acquisition of adventitious traits.

Visualizing Poxvirus Replication and Recombination Using Live-Cell Imaging.

A modernized version of an old saying goes that "If a picture is worth a thousand words, then a video is worth a million." Although made with reference to "YouTube", the quotation also has relevance for microbiologists when one considers how modern microscopes can be used to track biological fluorophores for hours without bleaching or phototoxicity. Confocal fluorescence microscopy provides a powerful tool for capturing dynamic processes within a cellular context that are better understood when viewed using time-lapse videos. In our laboratory we have long been interested in the links between poxvirus DNA replication and recombination and, since these are cytoplasmic viruses, such DNA-dependent processes are easily imaged throughout the virus life cycle without interference from signals coming from nuclear DNA. In this chapter we outline methods that can be used to follow the movement and replication of vaccinia virus DNA, and to also detect the products of poxvirus-catalyzed recombination reactions. We describe how to use the bacteriophage lambda DNA-binding protein, cro, as a way of labeling DNA within a cell when it is conjugated to fluorescent proteins. When used in conjunction with other fluorescent reagents, new labeling technologies, and tagged reporter constructs, these approaches can generate visually appealing and highly informative insights into diverse aspects of vaccinia virus biology.

Live-Cell Imaging of Vaccinia Virus Recombination.

Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren't detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories.