Identifying Protein Interactions with Viral DNA Genomes during Virus Infection.

Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.

Proliferating cell nuclear antigen inhibitors block distinct stages of herpes simplex virus infection.

Proliferating cell nuclear antigen (PCNA) forms a homotrimer that encircles replicating DNA and is bound by DNA polymerases to add processivity to cellular DNA synthesis. In addition, PCNA acts as a scaffold to recruit DNA repair and chromatin remodeling proteins to replicating DNA via its interdomain connecting loop (IDCL). Despite encoding a DNA polymerase processivity factor UL42, it was previously found that PCNA associates with herpes simplex virus type 1 (HSV-1) replication forks and is necessary for productive HSV-1 infection. To define the role that PCNA plays during viral DNA replication or a replication-coupled process, we investigated the effects that two mechanistically distinct PCNA inhibitors, PCNA-I1 and T2AA, have on the HSV-1 infectious cycle. PCNA-I1 binds at the interface between PCNA monomers, stabilizes the homotrimer, and may interfere with protein-protein interactions. T2AA inhibits select protein-protein interactions within the PCNA IDCL. Here we demonstrate that PCNA-I1 treatment results in reduced HSV-1 DNA replication, late gene expression, and virus production, while T2AA treatment results in reduced late viral gene expression and infectious virus production. To pinpoint the mechanisms by which PCNA inhibitors affect viral processes and protein recruitment to replicated viral DNA, we performed accelerated native isolation of proteins on nascent DNA (aniPOND). Results indicate that T2AA inhibits recruitment of the viral uracil glycosylase UL2 and transcription regulatory factors to viral DNA, likely leading to a defect in viral base excision repair and the observed defect in late viral gene expression and infectious virus production. In addition, PCNA-I1 treatment results in decreased association of the viral DNA polymerase UL30 and known PCNA-interacting proteins with viral DNA, consistent with the observed block in viral DNA replication and subsequent processes. Together, we conclude that inhibitors of cellular PCNA block recruitment of key viral and cellular factors to viral DNA to inhibit viral DNA synthesis and coupled processes.

HSV-1 DNA Replication-Coordinated Regulation by Viral and Cellular Factors.

DNA replication is an integral step in the herpes simplex virus type 1 (HSV-1) life cycle that is coordinated with the cellular DNA damage response, repair and recombination of the viral genome, and viral gene transcription. HSV-1 encodes its own DNA replication machinery, including an origin binding protein (UL9), single-stranded DNA binding protein (ICP8), DNA polymerase (UL30), processivity factor (UL42), and a helicase/primase complex (UL5/UL8/UL52). In addition, HSV-1 utilizes a combination of accessory viral and cellular factors to coordinate viral DNA replication with other viral and cellular processes. The purpose of this review is to outline the roles of viral and cellular proteins in HSV-1 DNA replication and replication-coupled processes, and to highlight how HSV-1 may modify and adapt cellular proteins to facilitate productive infection.

Limited carbon sources prevent sulfate remediation in circumneutral abandoned mine drainage.

Passive remediation systems (PRS) use both biotic and abiotic processes to precipitate contaminants from abandoned mine drainage (AMD) so that the contaminants do not spread into local watersheds. PRS are efficient at removing heavy metals but sulfate remediation frequently does not occur. To understand the reasons for the lack of sulfate remediation, we studied four PRS that treat circumneutral AMD and one raw mine drainage discharge. Using 16S sequencing analysis, microbial community composition revealed a high relative abundance of bacterial families with sulfur cycling genera. Anaerobic abiotic studies showed that sulfide was quickly geochemically oxidized in the presence of iron hydroxides, leading to a buildup of sulfur intermediates. Supplementation of laboratory grown microbes from the PRS with lactate demonstrated the ability of actively growing microbes to overcome this abiotic sulfide oxidation by increasing the rate of sulfate reduction. Thus, the lack of carbon sources in the PRS contributes to the lack of sulfate remediation. Bacterial community analysis of 16S rRNA gene revealed that while the microbial communities in different parts of the PRS were phylogenetically distinct, the contaminated environments selected for communities that shared similar metabolic capabilities.

Antiviral Properties of the LSD1 Inhibitor SP-2509.

Lysine-specific demethylase 1 (LSD1) targets cellular proteins, including histone H3, p53, E2F, and Dnmt1, and is involved in the regulation of gene expression, DNA replication, the cell cycle, and the DNA damage response. LSD1 catalyzes demethylation of histone H3K9 associated with herpes simplex virus 1 (HSV-1) immediate early (IE) promoters and is necessary for IE gene expression, viral DNA replication, and reactivation from latency. We previously found that LSD1 associates with HSV-1 replication forks and replicating viral DNA, suggesting that it may play a direct role in viral replication or coupled processes. We investigated the effects of the LSD1 inhibitor SP-2509 on the HSV-1 life cycle. Unlike previously investigated LSD1 inhibitors tranylcypromine (TCP) and OG-L002, which covalently attach to the LSD1 cofactor flavin adenine dinucleotide (FAD) to inhibit demethylase activity, SP-2509 has previously been shown to inhibit LSD1 protein-protein interactions. We found that SP-2509 does not inhibit HSV-1 IE gene expression or transcription factor and RNA polymerase II (Pol II) association with viral DNA prior to the onset of replication. However, SP-2509 does inhibit viral DNA replication, late gene expression, and virus production. We used EdC labeling of nascent viral DNA to image aberrant viral replication compartments that form in the presence of SP-2509. Treatment resulted in the formation of small replication foci that colocalize with replication proteins but are defective for Pol II recruitment. Taken together, these data highlight a potential new role for LSD1 in the regulation of HSV-1 DNA replication and gene expression after the onset of DNA replication.IMPORTANCE Treatment of HSV-1-infected cells with SP-2509 blocked viral DNA replication, gene expression after the onset of DNA replication, and virus production. These data support a potential new role for LSD1 in the regulation of viral DNA replication and successive steps in the virus life cycle, and further highlight the promising potential to utilize LSD1 inhibition as an antiviral approach.