Host-encoded Factors Research Articles

Targeting host deoxycytidine kinase mitigates Staphylococcus aureus abscess formation.

Host-directed therapy (HDT) is an emerging approach to overcome antimicrobial resistance in pathogenic microorganisms. Specifically, HDT targets host-encoded factors required for pathogen replication and survival without interfering with microbial growth or metabolism, thereby eliminating the risk of resistance development. By applying HDT and a drug repurposing approach, we demonstrate that (R)-DI-87, a clinical-stage anticancer drug and potent inhibitor of mammalian deoxycytidine kinase (dCK), mitigates Staphylococcus aureus abscess formation in organ tissues upon invasive bloodstream infection. Mechanistically, (R)-DI-87 shields phagocytes from staphylococcal death-effector deoxyribonucleosides that target dCK and the mammalian purine salvage pathway-apoptosis axis. In this manner, (R)-DI-87-mediated protection of immune cells amplifies macrophage infiltration into deep-seated abscesses, a phenomenon coupled with enhanced pathogen control, ameliorated immunopathology, and reduced disease severity. Thus, pharmaceutical blockade of dCK represents an advanced anti-infective intervention strategy against which staphylococci cannot develop resistance and may help to fight fatal infectious diseases in hospitalized patients.

Targeting host deoxycytidine kinase mitigates Staphylococcus aureus abscess formation

Host-directed therapy (HDT) is an emerging approach to overcome antimicrobial resistance in pathogenic microorganisms. Specifically, HDT targets host-encoded factors required for pathogen replication and survival without interfering with microbial growth or metabolism, thereby eliminating the risk of resistance development. By applying HDT and a drug repurposing approach, we demonstrate that (R)-DI-87, a clinical-stage anticancer drug and potent inhibitor of mammalian deoxycytidine kinase (dCK), mitigates Staphylococcus aureus abscess formation in organ tissues upon invasive bloodstream infection. Mechanistically, (R)-DI-87 shields phagocytes from staphylococcal death-effector deoxyribonucleosides that target dCK and the mammalian purine salvage pathway-apoptosis axis. In this manner, (R)-DI-87-mediated protection of immune cells amplifies macrophage infiltration into deep-seated abscesses, a phenomenon coupled with enhanced pathogen control, ameliorated immunopathology, and reduced disease severity. Thus, pharmaceutical blockade of dCK represents an advanced anti-infective intervention strategy against which staphylococci cannot develop resistance and may help to fight fatal infectious diseases in hospitalized patients.

Dynamic changes in Shiga toxin (Stx) 1 transducing phage throughout the evolution of O26:H11 Stx-producing Escherichia coli

Shiga toxin (Stx) is the key virulence factor of Stx-producing Escherichia coli (STEC). All known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages). Although the genetic diversity of Stx phages has frequently been described, systematic analyses of Stx phages in a single STEC lineage are limited. In this study, focusing on the O26:H11 STEC sequence type 21 (ST21) lineage, where the stx1a gene is highly conserved, we analysed the Stx1a phages in 39 strains representative of the entire ST21 lineage and found a high level of variation in Stx1a phage genomes caused by various mechanisms, including replacement by a different Stx1a phage at the same or different locus. The evolutionary timescale of events changing Stx1a phages in ST21 was also determined. Furthermore, by using an Stx1 quantification system developed in this study, we found notable variations in the efficiency of Stx1 production upon prophage induction, which sharply contrasted with the conserved iron regulated Stx1 production. These variations were associated with the Stx1a phage alteration in some cases but not in other cases; thus, Stx1 production in this STEC lineage was determined by differences not only in Stx1 phages but also in host-encoded factors.

Recipient UvrD helicase is involved in single- to double-stranded DNA conversion during conjugative plasmid transfer.

Dissemination of antibiotic resistance, a current societal challenge, is often driven by horizontal gene transfer through bacterial conjugation. During conjugative plasmid transfer, single-stranded (ss) DNA is transferred from the donor to the recipient cell. Subsequently, a complete double-stranded (ds) plasmid molecule is generated and plasmid-encoded genes are expressed, allowing successful establishment of the transconjugant cell. Such dynamics of transmission can be modulated by host- or plasmid-encoded factors, either in the donor or in the recipient cell. We applied transposon insertion sequencing to identify host-encoded factors that affect conjugative transfer frequency in Escherichia coli. Disruption of the recipient uvrD gene decreased the acquisition frequency of conjugative plasmids belonging to different incompatibility groups. Results from various UvrD mutants suggested that dsDNA binding activity and interaction with RNA polymerase are dispensable, but ATPase activity is required for successful plasmid establishment of transconjugant cells. Live-cell microscopic imaging showed that the newly transferred ssDNA within a uvrD- recipient often failed to be converted to dsDNA. Our work suggested that in addition to its role in maintaining genome integrity, UvrD is also key for the establishment of horizontally acquired plasmid DNA that drives genome diversity and evolution.

TFs for TEs: the transcription factor repertoire of mammalian transposable elements

Transposable elements (TEs) are genetic elements capable of changing position within the genome. Although their mobilization can constitute a threat to genome integrity, nearly half of modern mammalian genomes are composed of remnants of TE insertions. The first critical step for a successful transposition cycle is the generation of a full-length transcript. TEs have evolved cis-regulatory elements enabling them to recruit host-encoded factors driving their own, selfish transcription. TEs are generally transcriptionally silenced in somatic cells, and the mechanisms underlying their repression have been extensively studied. However, during germline formation, preimplantation development, and tumorigenesis, specific TE families are highly expressed. Understanding the molecular players at stake in these contexts is of utmost importance to establish the mechanisms regulating TEs, as well as the importance of their transcription to the biology of the host. Here, we review the transcription factors known to be involved in the sequence-specific recognition and transcriptional activation of specific TE families or subfamilies. We discuss the diversity of TE regulatory elements within mammalian genomes and highlight the importance of TE mobilization in the dispersal of transcription factor-binding sites over the course of evolution.

Extracellular vesicles mediate the horizontal transfer of an active LINE-1 retrotransposon

ABSTRACTLong interspersed element-1 (LINE-1 or L1) retrotransposons replicate through a copy-and-paste mechanism using an RNA intermediate. However, little is known about the physical transmission of retrotransposon RNA between cells. To examine the horizontal transfer of an active human L1 retrotransposon mediated by extracellular vesicles (EVs), human cancer cells were transfected with an expression construct containing a retrotransposition-competent human L1 tagged with a reporter gene. Using this model, active retrotransposition events were detected by screening for the expression of the reporter gene inserted into the host genome by retrotransposition. EVs including exosomes and microvesicles were isolated from cells by differential centrifugation. The enrichment of L1-derived reporter RNA transcripts were detected in EVs isolated from cells expressing active L1 retrotransposition. The delivery of reporter RNA was confirmed in recipient cells, and reporter genes were detected in the genome of recipient cells. Additionally, employing qRT-PCR, we found that host-encoded factors are activated in response to increased exposure to L1-derived RNA transcripts in recipient cells. Our results suggest that the horizontal transfer of retrotransposons can occur through the incorporation of RNA intermediates delivered via EVs and may have important implications for the intercellular regulation of gene expression and gene function.

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

Full-Length Glycosylated Gag of Murine Leukemia Virus Can Associate with the Viral Envelope as a Type I Integral Membrane Protein.

The glycosylated Gag protein (gPr80) of murine leukemia viruses (MLVs) has been shown to exhibit multiple roles in facilitating retrovirus release, infection, and resistance to host-encoded retroviral restriction factors, such as APOBEC3, SERINC3, and SERINC5. One way in which gPr80 helps MLVs to escape host innate immune restriction is by increasing capsid stability, a feature that protects viral replication intermediates from being detected by cytosolic DNA sensors. gPr80 also increases the resistance of MLVs to deamination and restriction by mouse APOBEC3 (mA3). How the gPr80 accessory protein, with its three N-linked glycosylation sites, contributes to these resistance mechanisms is still not fully understood. Here we further characterized the function of gPr80 and, more specifically, revealed that the asparagines targeted for glycosylation in gPr80 also contribute to capsid stability through their parallel involvement in the Pr65 Gag structural polyprotein. In fact, we demonstrate that sensitivity to deamination by the mA3 and human A3 proteins is directly linked to capsid stability. We also show that full-length gPr80 is detected in purified viruses. However, our results suggest that gPr80 is inserted in the NexoCcyto orientation of a type I integral membrane protein. Additionally, our experiments have revealed the existence of a large population of Env-deficient virus-like particles (VLPs) harboring gPr80 inserted in the opposite (NcytoCexo) polarity, which is typical of type II integral membrane proteins. Overall this study provides new insight into the complex nature of the MLV gPr80 accessory protein.IMPORTANCE Viruses have evolved numerous strategies to infect, spread in, and persist in their hosts. Here we analyze the details of how the MLV-encoded glycosylated Gag (gPr80) protein protects the virus from being restricted by host innate immune defenses. gPr80 is a variant of the structural Pr65 Gag protein with an 88-amino-acid extended leader sequence that directs the protein for translation and glycosylation in the endoplasmic reticulum. This study dissects the specific contributions of gPr80 glycans and capsid stability in helping the virus to infect cells, spread, and counteract the effects of the host intrinsic restriction factor APOBEC3. Overall this study provides further insight into the elusive role of the gPr80 protein.

Broad-Spectrum Inhibition of Respiratory Virus Infection by MicroRNA Mimics Targeting p38 MAPK Signaling.

The majority of antiviral therapeutics target conserved viral proteins, however, this approach confers selective pressure on the virus and increases the probability of antiviral drug resistance. An alternative therapeutic strategy is to target the host-encoded factors that are required for virus infection, thus minimizing the opportunity for viral mutations that escape drug activity. MicroRNAs (miRNAs) are small noncoding RNAs that play diverse roles in normal and disease biology, and they generally operate through the post-transcriptional regulation of mRNA targets. We have previously identified cellular miRNAs that have antiviral activity against a broad range of herpesvirus infections, and here we extend the antiviral profile of a number of these miRNAs against influenza and respiratory syncytial virus. From these screening experiments, we identified broad-spectrum antiviral miRNAs that caused >75% viral suppression in all strains tested, and we examined their mechanism of action using reverse-phase protein array analysis. Targets of lead candidates, miR-124, miR-24, and miR-744, were identified within the p38 mitogen-activated protein kinase (MAPK) signaling pathway, and this work identified MAPK-activated protein kinase 2 as a broad-spectrum antiviral target required for both influenza and respiratory syncytial virus (RSV) infection.

Transposon mutagenesis screen identifies host-encoded anti-viral factors

Abstract Ebola virus causes sporadic outbreaks of severe hemorrhagic fever. Owing to the high mortality rate, it poses a significant health risk and is a potential bioterrorism agent. However, our knowledge of how it usurps host cells to complete its life cycle, and more importantly, how this can be combated, is limited. Here we describe a genome-wide forward genetic approach to identify host genes, known as restriction factors, which confer resistance to viral infection. In this system cells are mutagenized with a transposon that activates genes close to its insertion site. Libraries of mutagenized cells are screened with a cytotoxic virus and resistant cell populations are selected. Illumina sequencing is used to characterize transposon insertion sites and identify candidate resistance genes. We have performed this screen in five human cell lines to identify genes conferring resistance to either vesicular stomatitis virus (VSV) or recombinant VSV bearing the Ebola virus surface glycoprotein. We have identified genes associated with resistance to only Ebola-VSV, or to both viruses, demonstrating that resistance occurs by targeting either viral entry or viral replication. One of the genes our screen identified is NPC1, the Ebola virus entry receptor, thus confirming that this is a valid method of identifying virus-host interactions. We have also identified a number of novel candidate Ebola restriction factors, which we are characterizing further, and are extending the screen to additional cell lines and viruses. This work provides new insights into host anti-viral defense mechanisms and may identify novel therapeutic targets that can be utilized to promote host-encoded anti-viral immunity.

Host restriction factors for hepatitis C virus.

Host-hepatitis C virus (HCV) interactions have both informed fundamental concepts of viral replication and pathogenesis and provided novel insights into host cell biology. These findings are illustrated by the recent discovery of host-encoded factors that restrict HCV infection. In this review, we briefly discuss these restriction factors in different steps of HCV infection. In each case, we discuss how these restriction factors were identified, the mechanisms by which they inhibit HCV infection and their potential contribution to viral pathogenesis.

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle

Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages.

Investigation of exponential RNA amplification by the Qβ replicase complex

Positive-stranded RNA viruses are common among human pathogenic viruses, which often cooperate with host proteins to fulfill essential functions during infection. One function is replication of the viral genome. The Qβ phage is a positive-stranded RNA virus that infects E.coli. The Qβ replicase holo enzyme comprises the phage-encoded RNA-dependent RNA polymerase (β-subunit) and the host-encoded translation elongation factors, EF-Ts and EF-Tu as well as the ribosomal protein S1. The Qβ replicase has an extraordinary ability to exponentially amplify RNA in vivo and in vitro. A prerequisite for this is release of product and template RNA as single strands that can serve as new templates in subsequent rounds of replication. The role of S1 in the Qβ replicase is not clear. Recently, S1 was found to promote release of single-stranded product in Qβ replicase–mediated RNA synthesis. We have undertaken NMR spectroscopy and crystallization trials to improve our understanding of distinct S1 domains in solution as well as the ribosome- and replicase-binding properties of S1. Expression of distinct S1 domains for NMR spectroscopy has been optimized by use of autoinduction and results in high yields of [13C15N]-labelled protein fragments. These have proven very suitable for NMR studies and spectra revealed both ordered and disordered regions in the protein. Studies are ongoing. The structure of the Qβ core complex was recently determined at 2.5Å resolution. Thus, co-crystallization of the Qβ core in complex with S1 domains was undertaken and different crystal forms were obtained. These initial crystals diffracted to 3.2Å resolution and data processing as well as further optimization of the crystals is ongoing. S1 is thought to bind the β-subunit close to a region lined with basic amino acids, which potentially could facilitate interactions with the template RNA backbone and split it from the product strand. We demonstrate that neutralization of these basic amino acids indeed decrease or abolish infectivity of the Qβ phage. However, only one mutation, R503A affects the exponential replication in vitro. Crystallization of the Qβ holo enzyme bound to a truncated legitimate RNA template will be the next step for investigation of the mechanism of exponential RNA amplification by Qβ replicase.

RstB2 Protein, the DNA Binding Protein of CTXΦ Phage from V. cholerae: Current Status and Pending Research

The phage-encoded protein RstB is required for CTXϕ phage integration, a lysogenic filamentous phage that contains the genes encoding cholera toxin, the main virulence factor of Vibrio cholerae. This mechanism of integration is catalyzed by the hostencoded tyrosine recombinases, XerC and XerD and some phage-encoded and host-encoded factors. This paper summarizes the results reported so far, of some bioinformatic and biochemical studies of the protein RSTB. Moreover, it has also been informed about ongoing bioinformatic studies which may contribute to clearing up the role of this protein in phage integration CTXϕ.

Resistance to simian immunodeficiency virus low dose rectal challenge is associated with higher constitutive TRIM5α expression in PBMC.

BackgroundAt least six host-encoded restriction factors (RFs), APOBEC3G, TRIM5α, tetherin, SAMHD1, schlafen 11, and Mx2 have now been shown to inhibit HIV and/or SIV replication in vitro. To determine their role in vivo in the resistance of macaques to mucosally-acquired SIV, we quantified both pre-exposure (basal) and post-exposure mRNA levels of these RFs, Mx1, and IFNγ in PBMC, lymph nodes, and duodenum of rhesus macaques undergoing weekly low dose rectal exposures to the primary isolate, SIV/DeltaB670.ResultsRepetitive challenge divided the monkeys into two groups with respect to their susceptibility to infection: highly susceptible (2–3 challenges, 5 monkeys) and poorly susceptible (≥6 challenges, 3 monkeys). Basal RF and Mx1 expression varied among the three tissues examined, with the lowest expression generally detected in duodenal tissues, and the highest observed in PBMC. The one exception was A3G whose basal expression was greatest in lymph nodes. Importantly, significantly higher basal expression of TRIM5α and Mx1 was observed in PBMC of animals more resistant to mucosal infection. Moreover, individual TRIM5α levels were stable throughout a year prior to infection. Post-exposure induction of these genes was also observed after virus appearance in plasma, with elevated levels in PBMC and duodenum transiently occurring 7–10 days post infection. They did not appear to have an effect on control of viremia. Interestingly, minimal to no induction was observed in the resistant animal that became an elite controller.ConclusionsThese results suggest that constitutively expressed TRIM5α appears to play a greater role in restricting mucosal transmission of SIV than that associated with type I interferon induction following virus entry. Surprisingly, this association was not observed with the other RFs. The higher basal expression of TRIM5α observed in PBMC than in duodenal tissues emphasizes the understated role of the second barrier to systemic infection involving the transport of virus from the mucosal compartment to the blood. Together, these observations provide a strong incentive for a more comprehensive examination of the intrinsic, variable control of constitutive expression of these genes in the sexual transmission of HIV.

Positive selection of primate genes that promote HIV-1 replication

Evolutionary analyses have revealed that most host-encoded restriction factors against HIV have experienced virus-driven selection during primate evolution. However, HIV also depends on the function of many human proteins, called host factors, for its replication. It is not clear whether virus-driven selection shapes the evolution of host factor genes to the extent that it is known to shape restriction factor genes. We show that five out of 40 HIV host factor genes (13%) analyzed do bear strong signatures of positive selection. Some of these genes (CD4, NUP153, RANBP2/NUP358) have been characterized with respect to the HIV lifecycle, while others (ANKRD30A/NY-BR-1 and MAP4) remain relatively uncharacterized. One of these, ANKRD30A, shows the most rapid evolution within this set of genes and is induced by interferon stimulation. We discuss how evolutionary analysis can aid the study of host factors for viral replication, just as it has the study of host immunity systems.

Expression profile of host restriction factors in HIV-1 elite controllers

BackgroundSeveral host-encoded antiviral factors suppress HIV-1 replication in a cell-autonomous fashion in vitro. The relevance of these defenses to the control of HIV-1 in vivo remains to be elucidated. We hypothesized that cellular restriction of HIV-1 replication plays a significant role in the observed suppression of HIV-1 in "elite controllers", individuals who maintain undetectable levels of viremia in the absence of antiretroviral therapy (ART). We comprehensively compared the expression levels of 34 host restriction factors and cellular activation levels in CD4+ T cells and sorted T cell subsets between elite controllers, HIV-1-infected (untreated) non-controllers, ART-suppressed, and uninfected individuals.ResultsExpression of schlafen 11, a codon usage-based inhibitor of HIV-1 protein synthesis, was significantly elevated in CD4+ T cells from elite controllers as compared to both non-controllers (p = 0.048) and ART-suppressed individuals (p = 0.024), with this effect most apparent in central memory CD4+ T cells. Schlafen 11 expression levels were comparable between controllers and uninfected individuals. Cumulative restriction factor expression was positively correlated with CD4+ T cell activation (r2 = 0.597, p < 0.0001), viral load (r2 = 0.34, p = 0.015), and expression of ISG15 (r2 = 0.73, p < 0.0001), a marker of interferon exposure. APOBEC3C, APOBEC3D, CTR9, TRIM26, and TRIM32 were elevated in elite controllers with respect to ART-suppressed individuals, while levels were comparable to uninfected individuals and non-controllers.ConclusionsHost restriction factor expression typically scales with cellular activation levels. However, the elevated mRNA and protein expression of schlafen 11, despite low activation and viral load, violates the global pattern and may be a signature characteristic of HIV-1 elite control.

Overexpression of Escherichia coli udk mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage

Successful infection of Escherichia coli by bacteriophage T7 relies upon the transcription of the T7 genome by two different RNA polymerases (RNAps). The bacterial RNAp transcribes early T7 promoters, whereas middle and late T7 genes are transcribed by the T7 RNAp. Gp2, a T7-encoded transcription factor, is a 7 kDa product of an essential middle T7 gene 2, and is a potent inhibitor of the host RNAp. The essential biological role of Gp2 is to inhibit transcription of early T7 genes that fail to terminate efficiently in order to facilitate the coordinated usage of the T7 genome by both host and phage RNAps. Overexpression of the E. coli udk gene, which encodes a uridine/cytidine kinase, interferes with T7 infection. We demonstrate that overexpression of udk antagonizes Gp2 function in E. coli in the absence of T7 infection and thus independently of T7-encoded factors. It seems that overexpression of udk reduces Gp2 stability and functionality during T7 infection, which consequently results in inadequate inhibition of host RNAp and in the accumulation of early T7 transcripts. In other words, overexpression of udk mimics the absence of Gp2 during T7 infection. Our study suggests that the transcriptional regulation of the T7 genome is surprisingly complex and might potentially be affected at many levels by phage- and host-encoded factors.

HIV Restriction Factors and Mechanisms of Evasion

Retroviruses have long been a fertile model for discovering host-pathogen interactions and their associated biological principles and processes. These advances have not only informed fundamental concepts of viral replication and pathogenesis but have also provided novel insights into host cell biology. This is illustrated by the recent descriptions of host-encoded restriction factors that can serve as effective inhibitors of retroviral replication. Here, we review our understanding of the three restriction factors that have been widely shown to be potent inhibitors of HIV-1: namely, APOBEC3G, TRIM5α, and tetherin. In each case, we discuss how these unrelated proteins were identified, the mechanisms by which they inhibit replication, the means used by HIV-1 to evade their action, and their potential contributions to viral pathogenesis as well as inter- and intraspecies transmission.

Expanding the role of Drosha to the regulation of viral gene expression

It is well-appreciated that viruses use host effectors for macromolecular synthesis and as regulators of viral gene expression. Viruses can encode their own regulators, but often use host-encoded factors to optimize replication. Here, we show that Drosha, an endoribonuclease best known for its role in the biogenesis of microRNAs (miRNAs), can also function to directly regulate viral gene expression. Kaposi's Sarcoma-associated Herpesvirus (KSHV) is associated with various tumors, and like all herpesviruses, has two modes of infection, latent and lytic, which are characterized by differential expression of viral gene products. Kaposin B (KapB) is a KSHV-encoded protein associated with cytokine production and cytotoxicity. We demonstrate that in addition to previously known transcriptional mechanisms, differences in Drosha levels contribute to low levels of KapB expression in latency and robust increases in expression during lytic replication. Thus, surprisingly, KSHV modulates Drosha activity differentially depending on the mode of replication. This regulation is dependent on Drosha-mediated cleavage, and KapB transcripts lacking the Drosha cleavage sites express higher levels of KapB, resulting in increased cell death. This work increases the known functions of Drosha and implies that tying viral gene expression to Drosha activity is advantageous for viruses.