Dysregulation of the miR-30c/DLL4 axis by circHIPK3 is essential for KSHV lytic replication.

Non-coding RNA (ncRNA) regulatory networks are emerging as critical regulators of gene expression. These intricate networks of ncRNA:ncRNA interactions modulate multiple cellular pathways and impact the development and progression of multiple diseases. Herpesviruses, including Kaposi's sarcoma-associated herpesvirus, are adept at utilising ncRNAs, encoding their own as well as dysregulating host ncRNAs to modulate virus gene expression and the host response to infection. Research has mainly focused on unidirectional ncRNA-mediated regulation of target protein-coding transcripts; however, we identify a novel host ncRNA regulatory network essential for KSHV lytic replication in B cells. KSHV-mediated upregulation of the host cell circRNA, circHIPK3, is a key component of this network, functioning as a competing endogenous RNA of miR-30c, leading to increased levels of the miR-30c target, DLL4. Dysregulation of this network highlights a novel mechanism of cell cycle control during KSHV lytic replication in B cells. Importantly, disruption at any point within this novel ncRNA regulatory network has a detrimental effect on KSHV lytic replication, highlighting the essential nature of this network and potential for therapeutic intervention.

M Segment-Based Minigenomes and Virus-Like Particle Assays as an Approach To Assess the Potential of Tick-Borne Phlebovirus Genome Reassortment.

Bunyaviruses have a tripartite negative-sense RNA genome. Due to the segmented nature of these viruses, if two closely related viruses coinfect the same host or vector cell, it is possible that RNA segments from either of the two parental viruses will be incorporated into progeny virions to give reassortant viruses. Little is known about the ability of tick-borne phleboviruses to reassort. The present study describes the development of minigenome assays for the tick-borne viruses Uukuniemi phlebovirus (UUKV) and Heartland phlebovirus (HRTV). We used these minigenome assays in conjunction with the existing minigenome system of severe fever with thrombocytopenia syndrome (SFTS) phlebovirus (SFTSV) to assess the abilities of viral N and L proteins to recognize, transcribe, and replicate the M segment-based minigenome of a heterologous virus. The highest minigenome activity was detected with the M segment-based minigenomes of cognate viruses. However, our findings indicate that several combinations utilizing N and L proteins of heterologous viruses resulted in M segment minigenome activity. This suggests that the M segment untranslated regions (UTRs) are recognized as functional promoters of transcription and replication by the N and L proteins of related viruses. Further, virus-like particle assays demonstrated that HRTV glycoproteins can package UUKV and SFTSV S and L segment-based minigenomes. Taken together, these results suggest that coinfection with these viruses could lead to the generation of viable reassortant progeny. Thus, the tools developed in this study could aid in understanding the role of genome reassortment in the evolution of these emerging pathogens in an experimental setting.IMPORTANCE In recent years, there has been a large expansion in the number of emerging tick-borne viruses that are assigned to the Phlebovirus genus. Bunyaviruses have a tripartite segmented genome, and infection of the same host cell by two closely related bunyaviruses can, in theory, result in eight progeny viruses with different genome segment combinations. We used genome analogues expressing reporter genes to assess the abilities of Phlebovirus nucleocapsid protein and RNA-dependent RNA polymerase to recognize the untranslated region of a genome segment of a related phlebovirus, and we used virus-like particle assays to assess whether viral glycoproteins can package genome analogues of related phleboviruses. Our results provide strong evidence that these emerging pathogens could reassort their genomes if they were to meet in nature in an infected host or vector. This reassortment process could result in viruses with new pathogenic properties.

Kv1.3-induced hyperpolarization is required for efficient Kaposi's sarcoma-associated herpesvirus lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus that is linked directly to the development of Kaposi's sarcoma. KSHV establishes a latent infection in B cells, which can be reactivated to initiate lytic replication, producing infectious virions. Using pharmacological and genetic silencing approaches, we showed that the voltage-gated K+ channel Kv1.3 in B cells enhanced KSHV lytic replication. The KSHV replication and transcription activator (RTA) protein increased the abundance of Kv1.3 and led to enhanced K+ channel activity and hyperpolarization of the B cell membrane. Enhanced Kv1.3 activity promoted intracellular Ca2+ influx, leading to the Ca2+-driven nuclear localization of KSHV RTA and host nuclear factor of activated T cells (NFAT) proteins and subsequently increased the expression of NFAT1 target genes. KSHV lytic replication and infectious virion production were inhibited by Kv1.3 blockers or silencing. These findings highlight Kv1.3 as a druggable host factor that is key to the successful completion of KSHV lytic replication.

Kaposi's sarcoma-associated herpesvirus induces specialised ribosomes to efficiently translate viral lytic mRNAs.

Historically, ribosomes were viewed as unchanged homogeneous macromolecular machines with no regulatory capacity for mRNA translation. An emerging concept is that heterogeneity of ribosomal composition exists, exerting a regulatory function or specificity in translational control. This is supported by recent discoveries identifying compositionally distinct specialised ribosomes that actively regulate mRNA translation. Viruses lack their own translational machinery and impose high translational demands on the host during replication. We explore the possibility that KSHV manipulates ribosome biogenesis producing specialised ribosomes which preferentially translate viral transcripts. Quantitative proteomic analysis identified changes in the stoichiometry and composition of precursor ribosomal complexes during the switch from latent to lytic replication. We demonstrate the enhanced association of ribosomal biogenesis factors BUD23 and NOC4L, and the KSHV ORF11 protein, with small ribosomal subunit precursor complexes during lytic replication. BUD23 depletion resulted in significantly reduced viral gene expression, culminating in dramatic reduction of infectious virion production. Ribosome profiling demonstrated BUD23 is essential for reduced association of ribosomes with KSHV uORFs in late lytic genes, required for the efficient translation of the downstream coding sequence. Results provide mechanistic insights into KSHV-mediated manipulation of cellular ribosome composition inducing a population of specialised ribosomes facilitating efficient translation of viral mRNAs.

m6A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication.

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Our results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection.

Mutational analysis of Rift Valley fever phlebovirus nucleocapsid protein indicates novel conserved, functional amino acids.

Rift Valley fever phlebovirus (RVFV; Phenuiviridae, Phlebovirus) is an important mosquito-borne pathogen of both humans and ruminants. The RVFV genome is composed of tripartite, single stranded, negative or ambisense RNAs. The small (S) segment encodes both the nucleocapsid protein (N) and the non-structural protein (NSs). The N protein is responsible for the formation of the viral ribonucleoprotein (RNP) complexes, which are essential in the virus life cycle and for the transcription and replication of the viral genome. There is currently limited knowledge surrounding the roles of the RVFV nucleocapsid protein in viral infection other than its key functions: N protein multimerisation, encapsidation of the RNA genome and interactions with the RNA-dependent RNA polymerase, L. By bioinformatic comparison of the N sequences of fourteen phleboviruses, mutational analysis, minigenome assays and packaging assays, we have further characterised the RVFV N protein. Amino acids P11 and F149 in RVFV N play an essential role in the function of RNPs and are neither associated with N protein multimerisation nor known nucleocapsid protein functions and may have additional roles in the virus life cycle. Amino acid Y30 exhibited increased minigenome activity despite reduced RNA binding capacity. Additionally, we have determined that the N-terminal arm of N protein is not involved in N-L interactions. Elucidating the fundamental processes that involve the nucleocapsid protein will add to our understanding of this important viral protein and may influence future studies in the development of novel antiviral strategies.

Characterization of the Zika virus induced small RNA response in Aedes aegypti cells.

RNA interference (RNAi) controls arbovirus infections in mosquitoes. Two different RNAi pathways are involved in antiviral responses: the PIWI-interacting RNA (piRNA) and exogenous short interfering RNA (exo-siRNA) pathways, which are characterized by the production of virus-derived small RNAs of 25-29 and 21 nucleotides, respectively. The exo-siRNA pathway is considered to be the key mosquito antiviral response mechanism. In Aedes aegypti-derived cells, Zika virus (ZIKV)-specific siRNAs were produced and loaded into the exo-siRNA pathway effector protein Argonaute 2 (Ago2); although the knockdown of Ago2 did not enhance virus replication. Enhanced ZIKV replication was observed in a Dcr2-knockout cell line suggesting that the exo-siRNA pathway is implicated in the antiviral response. Although ZIKV-specific piRNA-sized small RNAs were detected, these lacked the characteristic piRNA ping-pong signature motif and were bound to Ago3 but not Piwi5 or Piwi6. Silencing of PIWI proteins indicated that the knockdown of Ago3, Piwi5 or Piwi6 did not enhance ZIKV replication and only Piwi4 displayed antiviral activity. We also report that the expression of ZIKV capsid (C) protein amplified the replication of a reporter alphavirus; although, unlike yellow fever virus C protein, it does not inhibit the exo-siRNA pathway. Our findings elucidate ZIKV-mosquito RNAi interactions that are important for understanding its spread.