Human herpesviruses 6A/B (HHV-6A/B) can integrate their viral genomes in the telomeres of human chromosomes. The viral and cellular factors contributing to HHV-6A/B integration remain largely unknown, mostly due to the lack of efficient and reproducible cell culture models to study HHV-6A/B integration. In this study, we characterized the HHV-6A/B integration efficiencies in several human cell lines using two different approaches. First, after a short-term infection (5 h), cells were processed for single-cell cloning and analyzed for chromosomally integrated HHV-6A/B (ciHHV-6A/B). Second, cells were infected with HHV-6A/B and allowed to grow in bulk for 4 weeks or longer and then analyzed for the presence of ciHHV-6. Using quantitative PCR (qPCR), droplet digital PCR, and fluorescent in situ hybridization, we could demonstrate that HHV-6A/B integrated in most human cell lines tested, including telomerase-positive (HeLa, MCF-7, HCT-116, and HEK293T) and telomerase-negative cell lines (U2OS and GM847). Our results also indicate that inhibition of DNA replication, using phosphonoacetic acid, did not affect HHV-6A/B integration. Certain clones harboring ciHHV-6A/B spontaneously express viral genes and proteins. Treatment of cells with phorbol ester or histone deacetylase inhibitors triggered the expression of many viral genes, including U39, U90, and U100, without the production of infectious virus, suggesting that the tested stimuli were not sufficient to trigger full reactivation. In summary, both integration models yielded comparable results and should enable the identification of viral and cellular factors contributing to HHV-6A/B integration and the screening of drugs influencing viral gene expression, as well as the release of infectious HHV-6A/B from the integrated state.IMPORTANCE The analysis and understanding of HHV-6A/B genome integration into host DNA is currently limited due to the lack of reproducible and efficient viral integration systems. In the present study, we describe two quantitative cell culture viral integration systems. These systems can be used to define cellular and viral factors that play a role in HHV-6A/B integration. Furthermore, these systems will allow us to decipher the conditions resulting in virus gene expression and excision of the integrated viral genome resulting in reactivation.KEYWORDS chromosomal integration, ddPCR, telomere

A series of novel fluoroquinolone-Safirinium dye hybrids was synthesized by means of tandem Mannich-electrophilic amination reactions from profluorophoric isoxazolones and antibiotics bearing a secondary amino group at position 7 of the quinoline ring. The obtained fluorescent spiro fused conjugates incorporating quaternary nitrogen atoms were characterized by 1 H NMR, IR, MS, and elemental analysis. All the synthetic analogues (3a-h and 4a-h) were evaluated for their in vitro antimicrobial, bactericidal, and antibiofilm activities against a panel of Gram positive and Gram-negative pathogenic bacteria. The most active Safirinium Q derivatives of lomefloxacin (4d) and ciprofloxacin (4e) exhibited molar-based antibacterial activities comparable to the unmodified drugs and displayed considerable inhibitory potencies in E. coli DNA gyrase supercoiling assays with IC50 values in the low micromolar range. Zwiterionic hybrids were noticeably less lipophilic than the parent quinolones in micellar electrokinetic chromatography (MECK) experiments. The tests performed in the presence of phenylalanine-arginine β-naphthylamide (PAβN) or carbonyl cyanide m-chlorophenylhydrazone (CCCP) revealed that the conjugates are to some extent subject to bacterial efflux and cellular accumulation, respectively. Moreover, the hybrids did not exhibit notable cytotoxicity towards the HEK 293 control cell line and demonstrated low propensity for resistance development, as exemplified for compounds 3g and 4b. Finally, molecular docking experiments revealed that the synthesized compounds were able to bind in the fluoroquinolonebinding mode at S. aureus DNA gyrase and S. pneumoniae topoisomerase IV active sites.

Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency (P Ͻ 0.002); in contrast, BRACO-19 had no effect on HHV-6 integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affects the generation of cellular clones containing integrated HHV-6A.IMPORTANCE HHV-6A/B can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of various lengths (up to several kilobases) and end with a single-stranded 3= extension. To avoid recognition and induce a DNA damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure, referred to as a G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6A. This study is the first of its kind and suggests that telomerase activity is essential to restore a functional telomere of adequate length following HHV-6A integration.

Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3′ to 5′ exonuclease activity on dsDNA with a preference for 3′-recessed ends. Once the DNA strand reaches 8–10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3′ end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integration.

In an effort to find new repurposed antibacterial compounds, we performed the screening of an FDA-approved compounds library against Staphylococcus aureus American Type Culture Collection (ATCC) 25923. Compounds were evaluated for their capacity to prevent both planktonic growth and biofilm formation as well as to disrupt pre-formed biofilms. One of the identified initial hits was fingolimod (FTY720), an immunomodulator approved for the treatment of multiple sclerosis, which was then selected for follow-up studies. Fingolimod displayed a potent activity against S. aureus and S. epidermidis with a minimum inhibitory concentration (MIC) within the range of 12–15 µM at which concentration killing of all the bacteria was confirmed. A time–kill kinetic study revealed that fingolimod started to drastically reduce the viable bacterial count within two hours and we showed that no resistance developed against this compound for up to 20 days. Fingolimod also displayed a high activity against Acinetobacter baumannii (MIC 25 µM) as well as a modest activity against Escherichia coli and Pseudomonas aeruginosa. In addition, fingolimod inhibited quorum sensing in Chromobacterium violaceum and might therefore target this signaling pathway in certain Gram-negative bacteria. In conclusion, we present the identification of fingolimod from a compound library and its evaluation as a potential repurposed antibacterial compound.

Human herpesviruses 6A and 6B (HHV-6A/B) are unique among human herpesviruses in their ability to integrate their genome into host chromosomes. Viral integration occurs at the ends of chromosomes within the host telomeres. The ends of the HHV-6A/B genomes contain telomeric repeats that facilitate the integration process. Here, we report that productive infections are associated with a massive increase in telomeric sequences of viral origin. The majority of the viral telomeric signals can be detected within viral replication compartments (VRC) that contain the viral DNA processivity factor p41 and the viral immediate-early 2 (IE2) protein. Components of the shelterin protein complex present at telomeres, including TRF1 and TRF2 are also recruited to VRC during infection. Biochemical, immunofluorescence coupled with in situ hybridization and chromatin immunoprecipitation demonstrated the binding of TRF2 to the HHV-6A/B telomeric repeats. In addition, approximately 60% of the viral IE2 protein localize at cellular telomeres during infection. Transient knockdown of TRF2 resulted in greatly reduced (13%) localization of IE2 at cellular telomeres (p<0.0001). Lastly, TRF2 knockdown reduced HHV-6A/B integration frequency (p<0.05), while no effect was observed on the infection efficiency. Overall, our study identified that HHV-6A/B IE2 localizes to telomeres during infection and highlight the role of TRF2 in HHV-6A/B infection and chromosomal integration.

In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.

Bacterial biofilms are an important underlying cause for chronic infections. By switching into the biofilm state, bacteria can evade host defenses and withstand antibiotic chemotherapy. Despite the fact that biofilms at clinical and environmental settings are mostly composed of multiple microbial species, biofilm research has largely been focused on single-species biofilms. In this study, we investigated the interaction between two clinically relevant bacterial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) by label-free quantitative proteomics focusing on proteins associated with the bacterial cell surfaces (surfaceome) and proteins exported/released to the extracellular space (exoproteome). The changes observed in the surfaceome and exoproteome of P. aeruginosa pointed toward higher motility and lower pigment production when co-cultured with S. aureus. In S. aureus, lower abundances of proteins related to cell wall biosynthesis and cell division, suggesting increased persistence, were observed in the dual-species biofilm. Complementary phenotypic analyses confirmed the higher motility and the lower pigment production in P. aeruginosa when co-cultured with S. aureus. Higher antimicrobial tolerance associated with the co-culture setting was additionally observed in both species. To the best of our knowledge, this study is among the first systematic explorations providing insights into the dynamics of both the surfaceome and exoproteome of S. aureus and P. aeruginosa dual-species biofilms.