N 6-methyladenosine (m6A) and N6, 2′-O-Dimethyladenosine (m6Am) modifications (m6A/m) of messenger RNA mediate diverse cellular functions. Oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) has latent and lytic replication phases that are essential for the development of KSHV-associated cancers. To date, the role of m6A/m in KSHV replication and tumorigenesis is unclear. Here, we provide mechanistic insights by examining the viral and cellular m6A/m epitranscriptomes during KSHV latent and lytic infection. KSHV transcripts contain abundant m6A/m modifications during latent and lytic replication, and these modifications are highly conserved among different cell types and infection systems. Knockdown of YTHDF2 enhanced lytic replication by impeding KSHV RNA degradation. YTHDF2 binds to viral transcripts and differentially mediates their stability. KSHV latent infection induces 5′UTR hypomethylation and 3′UTR hypermethylation of the cellular epitranscriptome, regulating oncogenic and epithelial-mesenchymal transition pathways. KSHV lytic replication induces dynamic reprograming of epitranscriptome, regulating pathways that control lytic replication. These results reveal a critical role of m6A/m modifications in KSHV lifecycle and provide rich resources for future investigations.

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8 + T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

Discovered in 1994, KSHV is a human oncogenic gammaherpesvirus [1]. KSHV is causatively associated with several malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman's disease (MCD), and KSHV inflammatory cytokine syndrome (KICS), most of which are commonly found in HIV-1infected individuals [1,2,3,4]. KS is a multifocal mesenchymal neoplasm characterized by neo-angiogenesis, inflammatory infiltration, and spindle-shaped tumor cells that express mixed cellular markers, including vascular and lymphatic endothelial, mesenchymal, and hematopoietic precursor cells [5]. Early stage of KS primarily affects mucocutaneous tissues but advanced stage of KS is often involved with visceral organs [5]. KS is one of the most common malignancies in AIDS patients. While the advent of antiretroviral therapy has substantially reduced the incidence of KS in Western countries, it has stabilized or even rebound in recent years in some populations, and continues to be the most common cancer in some African regions [6]. Hence, KS remains to be one of the most important malignancies in AIDS patients causing significant morbidity and mortality.PEL is a rare and aggressive non-Hodgkin's B cell lymphoma clinically characterized by lymphomatous effusions in body cavities usually without tumor masses [7]. PEL often occurs in advanced AIDS patients with a decreased CD4 T cell count at diagnosis. Approximately, half of PEL patients have KS or are at risk for developing KS. PEL is resistant to conventional chemotherapy with a short median survival of less than 6 months [7].MCD is a polyclonal B cell lymphoproliferative disorder characterized by inflammatory symptoms, including fever, cachexia, lymphadenopathy, splenomegaly, cytopenia, and hypoalbuminemia [8]. MCD in the setting of HIV is typically associated with KSHV infection and is usually fatal without treatment. Furthermore, there is no established standard of treatment for KSHV-associated MCD [8].

N6-methyladenosine (m6A) is a dynamic post-transcriptional RNA modification influencing all aspects of mRNA biology. While m6A modifications during numerous viral infections have been described, the role of m6A in innate immune response remains unclear. Here, we examined cellular m6A epitranscriptomes during infections of Pseudomonas aeruginosa and herpes simplex virus type 1 (HSV-1), and lipopolysaccharide (LPS) stimulation to identify m6A-regulated innate immune response genes. We showed that a significant portion of cellular genes including many innate immune response genes underwent m6A modifications in 5'UTR and 3'UTR. We identified common and distinct m6A-modified genes under different stimulating conditions. Significantly, the expression of a subset of innate immune response genes was positively correlated with m6A level. Importantly, we identified genes that had significant enrichments of m6A peaks during P. aeruginosa infection following knockdown of m6A “eraser” ALKBH5, confirming the regulation of these genes by m6A and ALKBH5. Among them, we confirmed the association of m6A modification with gene expression in immune response genes TNFAIP3, IFIT1, IFIT2 and IFIH1. Taken together, our results revealed the vital role of m6A in regulating innate immunity against bacterial and viral infections. These works also provided rich resources for the scientific community.

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that regulate gene expression. They alter mRNA translation through base-pair complementarity, leading to regulation of genes during both physiological and pathological processes. Viruses have evolved mechanisms to take advantage of the host cells to multiply and/or persist over the lifetime of the host. Herpesviridae are a large family of double-stranded DNA viruses that are associated with a number of important diseases, including lymphoproliferative diseases. Herpesviruses establish lifelong latent infections through modulation of the interface between the virus and its host. A number of reports have identified miRNAs in a very large number of human and animal herpesviruses suggesting that these short non-coding transcripts could play essential roles in herpesvirus biology. This review will specifically focus on the recent advances on the functions of herpesvirus miRNAs in infection and pathogenesis.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus and the causative agent of varicella (chickenpox) in humans. Following primary infection, VZV establishes latency in the sensory ganglia and can reactivate to cause herpes zoster, more commonly known as shingles, which causes significant morbidity, and on rare occasions mortality, in the elderly. Because VZV infection is highly restricted to humans, the development of a reliable animal model has been challenging, and our understanding of VZV pathogenesis remains incomplete. As an alternative, infection of rhesus macaques with the homologous simian varicella virus (SVV) recapitulates the hallmarks of VZV infection and thus constitutes a robust animal model to provide critical insights into VZV pathogenesis and the host antiviral response. In this model, SVV infection results in the development of varicella during primary infection, generation of an adaptive immune response, establishment of latency in the sensory ganglia, and viral reactivation upon immune suppression. In this review, we discuss our current knowledge about host and viral factors involved in the establishment of SVV latency and reactivation as well as the important role played by T cells in SVV pathogenesis and antiviral immunity.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces malignant catarrhal fever (MCF), a fatal lymphoproliferative disease of ruminants, including cattle. The strain C500 has been cloned as an infectious, pathogenic bacterial artificial chromosome (BAC) that is used to study MCF. Although AlHV-1 infection can be established in cell culture, multiple passages in vitro cause a loss of virulence associated with rearrangements of the viral genome. Here, sequencing of the BAC clone showed that the long unique region (LUR) of the genome is nearly identical to that of the previously sequenced strain from which the BAC was derived, and identified the duplication and translocation of a region from within LUR, containing the entire coding sequences of ORF50-encoding reactivation transactivator Rta and A6-encoding bZIP protein genes. The duplicated region was further located to a position within the terminal repeat (TR) and its deletion resulted in lower ORF50 expression levels and reduced viral fitness. Finally, the presence of a similar but not identical duplication and translocation containing both genes was found in AlHV-1 strain WC11. These results indicate that selection pressure for enhanced viral fitness may drive the duplication of ORF50 and A6 in AlHV-1.

Viral semaphorins are semaphorin 7A (sema7A) mimics found in pox-and herpesviruses. Among herpesviruses, semaphorins are encoded by gammaherpesviruses of the Macavirus genus only. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus that persistently infects wildebeest asymptomatically but induces malignant catarrhal fever (MCF) when transmitted to several species of susceptible ruminants and the rabbit model. MCF is caused by the activation/proliferation of latently infected T lymphocytes. Viral semaphorins have been suggested to mediate immune evasion mechanisms and/or directly alter host T cell function. We studied AlHV-sema, the viral semaphorin encoded by the A3 gene of AlHV-1. Phylogenetic analyses revealed independent acquisition of pox-and herpesvirus semaphorins, suggesting that these proteins might have distinct functions. AlHV-sema showed a predicted three-dimensional structure very similar to sema7A and conserved key residues in sema7A-plexinC1 interaction. Expression analyses revealed that AlHV-sema is a secreted 93-kDa glycoprotein expressed during the early phase of virus replication. Purified AlHV-sema was able to bind to fibroblasts and dendritic cells and induce F-actin condensation and cell retraction through a plexinC1 and Rho/cofilin-dependent mechanism. Cytoskeleton rearrangement was further associated with inhibition of phagocytosis by dendritic cells and migration to the draining lymph node. Next, we generated recombinant viruses and demonstrated that the lack of A3 did not significantly affect virus growth in vitro and did not impair MCF induction and associated lymphoproliferative lesions. In conclusion, AlHV-sema has immune evasion functions through mechanisms similar to poxvirus semaphorin but is not directly involved in host T cell activation during MCF. IMPORTANCEWhereas most poxviruses encode viral semaphorins, semaphorin-like genes have only been identified in few gammaherpesviruses belonging to the Macavirus genus. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus carried asymptomatically by wildebeest but induces a latency-associated lymphoproliferative disease of T lymphocytes in various ruminant species, namely, malignant catarrhal fever (MCF). Viral semaphorins have been hypothesized to have immune evasion functions and/or be involved in activating latently infected T cells. We present evidence that the viral semaphorin AlHV-sema inhibits dendritic cell phagocytosis and migration to the draining lymph node, both being indispensable mechanisms for protective antiviral responses. Next, we engineered recombinant viruses unable to express AlHV-sema and demonstrated that this protein is dispensable for the induction of MCF. In conclusion, this study suggests that herpesvirus and poxvirus semaphorins have independently evolved similar functions to thwart the immune system of the host while AlHV-sema is not directly involved in MCF-associated T-cell activation. Semaphorins are members of a large family of secreted, membrane-anchored and transmembrane glycoproteins that can be f...