Molecular mechanisms of Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named “Rainbow-KSHV,” that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication. IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.

Studies on "HIT&RUN" effects by viral proteins are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled to identify both static and dynamic protein-protein interactions. Here we applied a PL method by generating recombinant Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) that suppress host interferon responses, and we examined KSHV vIRF-1 and vIRF-4 neighboring proteins to identify cellular proteins involved in innate immune regulation. PL identified 213 and 70 proteins as neighboring proteins of vIRF-1 and vIRF-4 during viral reactivation, and 47 proteins were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological roles such as mRNA processing and transcriptional regulation by TP53. Innate immune regulation by these commonly interacting 44 cellular proteins were examined by siRNAs and the splicing factor 3B family proteins were found to be associated with interferon transcription and act as suppressors of KSHV reactivation. We propose that recombinant mini-TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication, and selective splicing factors have a function in the regulation of innate immune responses. Importance Viral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to the use of affinity-precipitation approaches, the majority of studies focused on how the virus takes advantage of the newly-formed protein interactions for its own replication. Proximity labeling (PL) however, can also highlight transient and negative effects – those interactions which lead to dissociation from the existing protein complex. Here we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

Introduction: Encapsulating superoxide dismutase (SOD) in a semi-porous nanoparticle (NP) preserves myocardial function in rats following ischemia-reperfusion (I/R) injury. Cardiac targeting maximizes enzyme efficacy by enhancing retention. This study describes the formulation, characterization, and functional analysis of cardiac myosin (CM) targeted SOD NP. Method Targeted SOD NP with anti-CM antibody was developed in three steps and characterized with dynamic light scattering (DLS), colorimetric SOD assay, and in vitro imaging with isolated mature rat cardiomyocytes. In vivo function was assessed with a rat model of cardiac I/R injury in which the LAD was occluded for 60 minutes, ligation was released, and treatments were injected in the myocardium. Animals were recovered for 28 days and terminal pressure-volume (PV) loop analysis was performed. Result Anti-CM SOD NP was formulated by first encapsulating SOD in a polymersome containing an azide functional group. Second, a photoreactive antibody-binding domain was bonded to the azide. Third, covalent bonding of the NP to anti-CM antibody was performed with UV-light activation (Fig 1A). Consistent size was confirmed by DLS during formulation (Fig 1B). In vitro visualization of rhodamine-tagged anti-CM NP bound to mature cardiomyocytes confirmed antibody conjugation (Fig 1C). SOD activity was preserved during formulation and after degradation with Triton X, confirming conjugation did not affect porosity (Fig 1D). 53 rats underwent I/R injury and were randomized to 4 treatment groups of which 44 survived to terminal analysis (Fig 1E). Rats injected with anti-CM SOD NP had preserved EF (med 57%) and significant improvement in EF compared to PBS (med 40%, p=0.04). Conclusion Cardiac targeting of SOD NP with anti-CM antibody was successful and preserved EF in a rat I/R model. Ongoing future steps include correlation of scar volume with PV loop data, myocardial retention analysis and biodistribution of anti-CM NP.

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