Protein-RNA Interactome Analysis Reveals Wide Association of Kaposi's Sarcoma-Associated Herpesvirus ORF57 with Host Noncoding RNAs and Polysomes.

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding posttranscriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57 cross-linking immunoprecipitation (CLIP) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIP RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host noncoding and protein-coding mRNAs. We found that ORF57 binds and regulates expression of a subset of host long noncoding RNAs (lncRNAs), including LINC00324, LINC00355, and LINC00839, which are involved in cell growth. ORF57 binds small nucleolar RNAs (snoRNAs) responsible for 18S and 28S rRNA modifications but does not interact with fibrillarin or NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57 RNA immunoprecipitation (RIP)-snoRNA array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57 RIP and Northern blot analyses using a 32P-labeled oligonucleotide probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligonucleotides from the rRNA regions that ORF57 binds for oligonucleotide pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and that its association with polysomes increases PABPC1 binding to polysomes but prevents Ago2 association with polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. IMPORTANCE As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus-infected cells. In this study, ORF57 was found to interact with many host noncoding RNAs, including lncRNAs, snoRNAs, and rRNAs, to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the RNA motifs identified by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins and with rRNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4 but does not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC1 but prevents Ago2 association with polysomes. Data provide compelling evidence on how ORF57 in KSHV-infected cells might regulate protein synthesis by blocking Ago2's hostile activities on translation.

KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182.

Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.

KSHV inhibits stress granule formation by viral ORF57 blocking PKR activation.

TIA-1 positive stress granules (SG) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi's sarcoma-associated herpesvirus (KSHV) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT) and protein kinase R (PKR) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection.

Cell Type- and Tissue Context-dependent Nuclear Distribution of Human Ago2.

Argonaute-2 protein (Ago2), a major component of RNA-induced silencing complex (RISC), has been viewed as a cytoplasmic protein. In this study, we demonstrated by immunofluorescence confocal microscopy that Ago2 is distributed mainly as a nuclear protein in primary human foreskin keratinocytes in monolayer cultures and their derived organotypic (raft) cultures, although it exhibits only a minimal level of nuclear distribution in continuous cell lines such as HeLa and HaCaT cells. Oncogenic human papillomavirus type 16 (HPV16) or type 18 (HPV18) infection of the keratinocytes does not affect the nuclear Ago2 distribution. Examination of human tissues reveals that Ago2 exhibits primarily as a nuclear protein in skin, normal cervix, and cervical cancer tissues, but not in larynx. Together, our data provide the first convincing evidence that the subcellular distribution of Ago2 occurs in a cell type- and tissue context-dependent manner and may correlate with its various functions in regulation of gene expression.

Hepatitis C virus is primed by CD81 protein for low pH-dependent fusion.

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.

Receptor tyrosine kinase inhibitors that block replication of influenza a and other viruses.

We have previously reported that two receptor tyrosine kinase inhibitors (RTKIs), called AG879 and tyrphostin A9 (A9), can each block the replication of influenza A virus in cultured cells. In this study, we further characterized the in vitro antiviral efficacies and specificities of these agents. The 50% effective concentration (EC(50)) of each against influenza A was found to be in the high nanomolar range, and the selectivity index (SI = 50% cytotoxic concentration [CC(50)]/EC(50)) was determined to be >324 for AG879 and 50 for A9, indicating that therapeutically useful concentrations of each drug produce only low levels of cytotoxicity. Each compound showed efficacy against representative laboratory strains of both human influenza A (H1N1 or H3N2) and influenza B viruses. Importantly, no drug-resistant influenza virus strains emerged even after 25 viral passages in the presence of AG879, whereas viruses resistant to amantadine appeared after only 3 passages. AG879 and A9 each also exhibited potent inhibitory activity against a variety of other RNA and DNA viruses, including Sendai virus (Paramyxoviridae), herpes simplex virus (Herpesviridae), mouse hepatitis virus (Coronaviridae), and rhesus rotavirus (Reoviridae), but not against Pichinde virus (Arenaviridae). These results together suggest that RTKIs may be useful as therapeutics against viral pathogens, including but not limited to influenza, due to their high selectivity indices, low frequency of drug resistance, and broad-spectrum antiviral activities.

Reciprocal regulation of AKT and MAP kinase dictates virus-host cell fusion.

Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy.

RNA Granules in Antiviral Innate Immunity: A Kaposi's Sarcoma-Associated Herpesvirus Journey.

RNA granules are cytoplasmic, non-membranous ribonucleoprotein compartments that form ubiquitously and are often referred to as foci for post-transcriptional gene regulation. Recent research on RNA processing bodies (PB) and stress granules (SG) has shown wide implications of these cytoplasmic RNA granules and their components in suppression of RNA translation as host intracellular innate immunity against infecting viruses. Many RNA viruses either counteract or co-opt these RNA granules; however, many fundamental questions about DNA viruses with respect to their interaction with these two RNA granules remain elusive. Kaposi's sarcoma-associated herpesvirus (KSHV), a tumor-causing DNA virus, exhibits two distinct phases of infection and encodes ∼90 viral gene products during the lytic phase of infection compared to only a few (∼5) during the latent phase. Thus, productive KSHV infection relies heavily on the host cell translational machinery, which often links to the formation of PB and SG. One major question is how KSHV counteracts the hostile environment of RNA granules for its productive infection. Recent studies demonstrated that KSHV copes with the translational suppression by cellular RNA granules, PB and SG, by expressing ORF57, a viral RNA-binding protein, during KSHV lytic infection. ORF57 interacts with Ago2 and GW182, two major components of PB, and prevents the scaffolding activity of GW182 at the initial stage of PB formation in the infected cells. ORF57 also interacts with protein kinase R (PKR) and PKR-activating protein (PACT) to block PKR dimerization and kinase activation, and thus inhibits eIF2α phosphorylation and SG formation. The homologous immediate-early regulatory protein ICP27 of herpes simplex virus type 1 (HSV-1), but not the EB2 protein of Epstein-Barr virus (EBV), shares this conserved inhibitory function with KSHV ORF57 on PB and SG. Through KSHV ORF57 studies, we have learned much about how a DNA virus in the infected cells is equipped to evade host antiviral immunity for its replication and productive infection. KSHV ORF57 would be an excellent viral target for development of anti-KSHV-specific therapy.

Analysis of the dark proteome of Chandipura virus reveals maximum propensity for intrinsic disorder in phosphoprotein.

Chandipura virus (CHPV, a member of the Rhabdoviridae family) is an emerging pathogen that causes rapidly progressing influenza-like illness and acute encephalitis often leading to coma and death of the human host. Given several CHPV outbreaks in Indian sub-continent, recurring sporadic cases, neurological manifestation, and high mortality rate of this infection, CHPV is gaining global attention. The 'dark proteome' includes the whole proteome with special emphasis on intrinsically disordered proteins (IDP) and IDP regions (IDPR), which are proteins or protein regions that lack unique (or ordered) three-dimensional structures within the cellular milieu. These proteins/regions, however, play a number of vital roles in various biological processes, such as cell cycle regulation, control of signaling pathways, etc. and, therefore, are implicated in many human diseases. IDPs and IPPRs are also abundantly found in many viral proteins enabling their multifunctional roles in the viral life cycles and their capability to highjack various host systems. The unknown abundance of IDP and IDPR in CHPV, therefore, prompted us to analyze the dark proteome of this virus. Our analysis revealed a varying degree of disorder in all five CHPV proteins, with the maximum level of intrinsic disorder propensity being found in Phosphoprotein (P). We have also shown the flexibility of P protein using extensive molecular dynamics simulations up to 500 ns (ns). Furthermore, our analysis also showed the abundant presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs) in CHPV proteins. The identification of IDPs/IDPRs in CHPV proteins suggests that their disordered regions may function as potential interacting domains and may also serve as novel targets for disorder-based drug designs.

Viral Regulation of RNA Granules in Infected Cells.

RNA granules are cytoplasmic, microscopically visible, non-membrane ribo-nucleoprotein structures and are important posttranscriptional regulators in gene expression by controlling RNA translation and stability. TIA/G3BP/PABP-specific stress granules (SG) and GW182/DCP-specific RNA processing bodies (PB) are two major distinguishable RNA granules in somatic cells and contain various ribosomal subunits, translation factors, scaffold proteins, RNA-binding proteins, RNA decay enzymes and helicases to exclude mRNAs from the cellular active translational pool. Although SG formation is inducible due to cellular stress, PB exist physiologically in every cell. Both RNA granules are important components of the host antiviral defense. Virus infection imposes stress on host cells and thus induces SG formation. However, both RNA and DNA viruses must confront the hostile environment of host innate immunity and apply various strategies to block the formation of SG and PB for their effective infection and multiplication. This review summarizes the current research development in the field and the mechanisms of how individual viruses suppress the formation of host SG and PB for virus production.

Site-specific phosphorylation of villin remodels the actin cytoskeleton to regulate Sendai viral glycoprotein-mediated membrane fusion.

Connivance of cellular factors during virus-host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin-null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c-src. However, the villin RRI mutant, lacking actin-severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome-CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell-type dependent phosphorylation in regulating membrane fusion.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis-acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors.IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter.