Conserved linear motif within the immediate early protein ORF45 promotes its engagement with KSHV lytic cycle-promoting forkhead transcription factors, FOXK1 and FOXK2.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism.IMPORTANCEThe dysregulation of Forkhead transcription factors contributes to many different human diseases, including cancers, but their impact on herpesvirus lifecycle and pathogenesis is less understood. Our study uncovers a critical pro-lytic function of the FOXK subfamily and its requirement for KSHV lytic reactivation in PEL. We found that FOXK proteins bind to a key immediate early KSHV protein ORF45 using its novel short linear S/T motif. Notably, even though ORF45 homologs in gammaherpesviruses are highly diverse, we identified a similar S/T short linear motif in ORF45 homologs and also showed an evolutionary conserved interaction between FOXK proteins and ORF45 homologs of MHV68 and RRV. Our study provides a basis for future studies in animal models to evaluate the role of FOXK proteins and the impact of their interactions with ORF45 in gammaherpesvirus infection and pathogenesis. Targeting these interactions could allow a novel way to limit gammaherpesvirus infections.

Genome-Wide Transcriptional Roles of KSHV Viral Interferon Regulatory Factors in Oral Epithelial Cells.

The viral interferon regulatory factors (vIRFs) of KSHV are known to dysregulate cell signaling pathways to promote viral oncogenesis and to block antiviral immune responses to facilitate infection. However, it remains unknown to what extent each vIRF plays a role in gene regulation. To address this, we performed a comparative analysis of the protein structures and gene regulation of the four vIRFs. Our structure prediction analysis revealed that despite their low amino acid sequence similarity, vIRFs exhibit high structural homology in both their DNA-binding domain (DBD) and IRF association domain. However, despite this shared structural homology, we demonstrate that each vIRF regulates a distinct set of KSHV gene promoters and human genes in epithelial cells. We also found that the DBD of vIRF1 is essential in regulating the expression of its target genes. We propose that the structurally similar vIRFs evolved to possess specialized transcriptional functions to regulate specific genes.

Kaposi's Sarcoma-Associated Herpesvirus Immediate Early Proteins Trigger FOXQ1 Expression in Oral Epithelial Cells, Engaging in a Novel Lytic Cycle-Sustaining Positive Feedback Loop.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that can replicate in oral epithelial cells to promote viral transmission via saliva. To identify novel regulators of KSHV oral infection, we performed a transcriptome analysis of KSHV-infected primary human gingival epithelial (HGEP) cells, which identified the gene coding for the host transcription factor FOXQ1 as the top induced host gene. FOXQ1 is nearly undetectable in uninfected HGEP and telomerase-immortalized gingival keratinocytes (TIGK) cells but is highly expressed within hours of KSHV infection. We found that while the FOXQ1 promoter lacks activating histone acetylation marks in uninfected oral epithelial cells, these marks accumulate in the FOXQ1 promoter in infected cells, revealing a rapid epigenetic reprogramming event. To evaluate FOXQ1 function, we depleted FOXQ1 in KSHV-infected TIGK cells, which resulted in reduced accumulation of KSHV lytic proteins and viral DNA over the course of 4 days of infection, uncovering a novel lytic cycle-sustaining role of FOXQ1. A screen of KSHV lytic proteins demonstrated that the immediate early proteins ORF45 and replication and transcription activator (RTA) were both sufficient for FOXQ1 induction in oral epithelial cells, indicating active involvement of incoming and rapidly expressed factors in altering host gene expression. ORF45 is known to sustain extracellular signal-regulated kinase (ERK) p90 ribosomal s6 kinase (RSK) pathway activity to promote lytic infection. We found that an ORF45 mutant lacking RSK activation function failed to induce FOXQ1 in TIGK cells, revealing that ORF45 uses a shared mechanism to rapidly induce both host and viral genes to sustain lytic infection in oral epithelial cells. IMPORTANCE The oral cavity is a primary site of initial contact and entry for many viruses. Viral replication in the oral epithelium promotes viral shedding in saliva, allowing interpersonal transmission, as well as spread to other cell types, where chronic infection can be established. Understanding the regulation of KSHV infection in the oral epithelium would allow for the design of universal strategies to target the first stage of viral infection, thereby halting systemic viral pathogenesis. Overall, we uncover a novel positive feedback loop in which immediate early KSHV factors drive rapid host reprogramming of oral epithelial cells to sustain the lytic cycle in the oral cavity.

The ORF45 Protein of Kaposi's Sarcoma-Associated Herpesvirus and Its Critical Role in the Viral Life Cycle.

Kaposi's sarcoma-associated herpesvirus (KSHV) protein ORF45 is a virion-associated tegument protein that is unique to the gammaherpesvirus family. Generation of KSHV ORF45-knockout mutants and their subsequent functional analyses have permitted a better understanding of ORF45 and its context-specific and vital role in the KSHV lytic cycle. ORF45 is a multifaceted protein that promotes infection at both the early and late phases of the viral life cycle. As an immediate-early protein, ORF45 is expressed within hours of KSHV lytic reactivation and plays an essential role in promoting the lytic cycle, using multiple mechanisms, including inhibition of the host interferon response. As a tegument protein, ORF45 is necessary for the proper targeting of the viral capsid for envelopment and release, affecting the late stage of the viral life cycle. A growing list of ORF45 interaction partners have been identified, with one of the most well-characterized being the association of ORF45 with the host extracellular-regulated kinase (ERK) p90 ribosomal s6 kinase (RSK) signaling cascade. In this review, we describe ORF45 expression kinetics, as well as the host and viral interaction partners of ORF45 and the significance of these interactions in KSHV biology. Finally, we discuss the role of ORF45 homologs in gammaherpesvirus infections.

Clinical Manifestations and Epigenetic Regulation of Oral Herpesvirus Infections.

The oral cavity is often the first site where viruses interact with the human body. The oral epithelium is a major site of viral entry, replication and spread to other cell types, where chronic infection can be established. In addition, saliva has been shown as a primary route of person-to-person transmission for many viruses. From a clinical perspective, viral infection can lead to several oral manifestations, ranging from common intraoral lesions to tumors. Despite the clinical and biological relevance of initial oral infection, little is known about the mechanism of regulation of the viral life cycle in the oral cavity. Several viruses utilize host epigenetic machinery to promote their own life cycle. Importantly, viral hijacking of host chromatin-modifying enzymes can also lead to the dysregulation of host factors and in the case of oncogenic viruses may ultimately play a role in promoting tumorigenesis. Given the known roles of epigenetic regulation of viral infection, epigenetic-targeted antiviral therapy has been recently explored as a therapeutic option for chronic viral infection. In this review, we highlight three herpesviruses with known roles in oral infection, including herpes simplex virus type 1, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. We focus on the respective oral clinical manifestations of these viruses and their epigenetic regulation, with a specific emphasis on the viral life cycle in the oral epithelium.

Early Initiation of Breastfeeding Among Maya Mothers in the Western Highlands of Guatemala: Practices and Beliefs.

BACKGROUND: Guatemala exhibits the sixth highest rate of child stunting worldwide, and stunting disproportionately affects Guatemala indigenous communities. In a country struggling to combat this result of malnutrition, early child nutrition is especially critical. Specifically, early initiation of breastfeeding is important for the development of newborn infants. Understanding beliefs and practices related to early initiation of breastfeeding in Maya Guatemala may provide an avenue to guide nutrition interventions in indigenous communities. Research aim: This study aimed to determine major beliefs and practices associated with early initiation of breastfeeding among Maya mothers in Lake Atitlán, Guatemala. METHODS: As part of a larger study to assess child nutrition in the Lake Atitlán region, we created a series of semistructured interview questions to document breastfeeding practices and beliefs among mothers. We conducted and audio-recorded in-person interviews that were translated from Kaqchikel, the local language, to Spanish by a community assistant. RESULTS: We conducted 178 interviews with mothers; 76% practiced early initiation. Early initiation was associated with the village and complementary feeding practices. Mothers held a variety of beliefs about the value of colostrum, and these beliefs were associated with the village. Mothers who held negative beliefs toward colostrum were more likely to delay breastfeeding initiation. CONCLUSION: Although most Maya mothers practice early initiation, the intervillage disparity in breastfeeding practices demonstrates a need to geographically focus breastfeeding interventions. Our novel insights into the breastfeeding beliefs among Maya mothers will serve as a guide to structure culturally competent breastfeeding education interventions in indigenous communities.

The Physician Recommendation Coding System (PhyReCS): A Reliable and Valid Method to Quantify the Strength of Physician Recommendations During Clinical Encounters.

BACKGROUND: Physicians' recommendations affect patients' treatment choices. However, most research relies on physicians' or patients' retrospective reports of recommendations, which offer a limited perspective and have limitations such as recall bias. OBJECTIVE: To develop a reliable and valid method to measure the strength of physician recommendations using direct observation of clinical encounters. METHODS: Clinical encounters (n = 257) were recorded as part of a larger study of prostate cancer decision making. We used an iterative process to create the 5-point Physician Recommendation Coding System (PhyReCS). To determine reliability, research assistants double-coded 50 transcripts. To establish content validity, we used 1-way analyses of variance to determine whether relative treatment recommendation scores differed as a function of which treatment patients received. To establish concurrent validity, we examined whether patients' perceived treatment recommendations matched our coded recommendations. RESULTS: The PhyReCS was highly reliable (Krippendorf's alpha = 0.89, 95% CI [0.86, 0.91]). The average relative treatment recommendation score for each treatment was higher for individuals who received that particular treatment. For example, the average relative surgery recommendation score was higher for individuals who received surgery versus radiation (mean difference = 0.98, SE = 0.18, P < 0.001) or active surveillance (mean difference = 1.10, SE = 0.14, P < 0.001). Patients' perceived recommendations matched coded recommendations 81% of the time. CONCLUSION: The PhyReCS is a reliable and valid way to capture the strength of physician recommendations. We believe that the PhyReCS would be helpful for other researchers who wish to study physician recommendations, an important part of patient decision making.