Several lines of evidence suggest that recombination plays a central role in replication and evolution of herpes simplex virus-1 (HSV-1). G-quadruplex (G4)-motifs have been linked to recombination events in human and microbial genomes, but their role in recombination has not been studied in DNA viruses. The availability of near full-length sequences from 40 HSV-1 recombinant strains with exact position of the recombination breakpoints provided us with a unique opportunity to investigate the role of G4-motifs in recombination among herpes viruses. We mapped the G4-motifs in the parental and all the 40 recombinant strains. Interestingly, the genome-wide distribution of breakpoints closely mirrors the G4 densities in the HSV-1 genome; regions of the genome with higher G4 densities had higher number of recombination breakpoints. Biophysical characterization of oligonucleotides from a subset of predicted G4-motifs confirmed the formation of G-quadruplex structures. Our analysis also reveals that G4-motifs are enriched in regions flanking the recombination breakpoints. Interestingly, about 11% of breakpoints lie within a G4-motif, making these DNA secondary structures hotspots for recombination in the HSV-1 genome. Breakpoints within G4-motifs predominantly lie within G4-clusters rather than individual G4-motifs. Of note, we identified the terminal guanosine of G4-clusters at the boundaries of the UL (unique long) region on either side of the OriL (origin of replication within UL) represented the commonest breakpoint among the HSV-1 recombinants. Our findings suggest a correlation between the HSV-1 recombination landscape and the distribution of G4-motifs and G4-clusters, with possible implications for the evolution of DNA viruses.

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV. IMPORTANCE: Inositol hexakisphosphate (IP6) is crucial for the assembly and replication of HIV-1. IP6 is packaged in HIV-1 particles and stabilizes the viral core enabling it to synthesize viral DNA early in viral infection. While its importance for HIV-1 is well established, its significance for other retroviruses is unknown. Here we report the role of IP6 in the gammaretrovirus, murine leukemia virus (MLV). We found that like HIV-1, MLV packages IP6, and as in HIV-1, IP6 stabilizes the MLV core thus promoting reverse transcription. Interestingly, we discovered a key difference in the role of IP6 in MLV versus HIV-1: while HIV-1 is not dependent upon IP6 levels in target cells, MLV replication is significantly reduced in IP6-deficient cell lines. We suggest that this difference in IP6 requirements reflects key differences between HIV-1 and MLV replication.

Pseudovirus-based assays are efficient and accessible platforms for measuring neutralizing antibody titers and screening antiviral drugs against enveloped viruses. They typically employ two types of packaging systems, retroviral-based or vesicular stomatitis virus (VSV)-based systems. These systems have been validated for several high-risk pathogens, including Ebola, Marburg, Dengue and, more recently, SARS-CoV-2, as they offer a safe alternative for studying high-containment viruses. In a recent study (Wu X, Liu F, Li T, Li D, Shen Y, Zhang X, Liu S, Jiang Q, Zhao C, Nie J, Wang Y, Feng B, Liu W, Huang W, Microbiol Spectr, 13:e01557-24, 2025, https://doi.org/10.1128/spectrum.01557-24 ), Wu and colleagues developed a pseudovirus system and optimized it for neutralization assays targeting the newly identified Dehong virus (DEHV). The authors also utilized this system to detect bioluminescent signals in mouse models. Importantly, using this pseudovirus platform, they confirmed that Niemann-Pick C1 (NPC1) functions as the entry receptor for DEHV, consistent with prior findings using infectious viruses. This work provides valuable insights into the limited understanding of DEHV biology.

GS-441524, the parent nucleoside of remdesivir, engages the SARS-CoV-2 macrodomain (Mac1) in cells, and its diphosphate metabolite exhibits enhanced inhibitory activity.