Comprehensive Identification of Host Modulators of HIV-1 Replication using Multiple Orthologous RNAi Reagents

SummaryRNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene’s phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1–host cell interactions

IFITM3 Inhibits Influenza A Virus Infection by Preventing Cytosolic Entry

To replicate, viruses must gain access to the host cell's resources. Interferon (IFN) regulates the actions of a large complement of interferon effector genes (IEGs) that prevent viral replication. The interferon inducible transmembrane protein family members, IFITM1, 2 and 3, are IEGs required for inhibition of influenza A virus, dengue virus, and West Nile virus replication in vitro. Here we report that IFN prevents emergence of viral genomes from the endosomal pathway, and that IFITM3 is both necessary and sufficient for this function. Notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by IFN, and IFITM3 was required and sufficient for this action. We further demonstrate that IFN expands Rab7 and LAMP1-containing structures, and that IFITM3 overexpression is sufficient for this phenotype. Moreover, IFITM3 partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. Collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to IFITM3's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. Multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by IFN. Therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats

Ebola Virus VP30 Is an RNA Binding Protein▿

The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA

IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages

Summary: Activation of the TLR4 signaling pathway by lipopolysaccharide (LPS) leads to induction of both inflammatory and interferon-stimulated genes, but the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remain poorly understood. In a genome-wide small interfering RNA (siRNA) screen of the LPS-induced tumor necrosis factor α (TNF-α) response in macrophages, we identify the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identifies a positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of LPS-induced gene classes. We demonstrate that these effects of IFIT1 are mediated through modulation of a Sin3A-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate a function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program. : John et al. describe a function for IFIT1 in the innate immune response. Previously considered an antiviral protein, IFIT1 is identified as a reciprocal modulator of bacterially induced pro-inflammatory and interferon genes and shown to associate with chromatin regulators to modulate transcription and the host response to bacterial infection. Keywords: interferon, IFIT1, TLR4, interferon gene program, pro-inflammatory gene program, RNAi screen, TNF-

Inhibition of Tip60 Reduces Lytic and Latent Gene Expression of Kaposi’s Sarcoma-Associated Herpes Virus (KSHV) and Proliferation of KSHV-Infected Tumor Cells

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus responsible for the development of Kaposi’s sarcoma, primary effusion lymphoma (PEL), and Multicentric Castleman’s disease in immunocompromised individuals. Despite the burden of these diseases there are few treatment options for afflicted individuals, due in part to our limited understanding of virus-host interactions. Tip60, a histone aceytltransferase (HAT) has been previously shown to interact with both the KSHV latency associated nuclear antigen protein (LANA), which is the main factor in maintaining the viral latent state, and ORF36, a viral kinase expressed in the lytic phase. We further investigated Tip60-virus interaction to ascertain Tip60’s role in the viral life cycle and its potential as a target for future therapeutics. Through modulation of Tip60 expression in HEK293T cells harboring a plasmid containing the KSHV viral episome, Bac36, we found that Tip60 is vital for both lytic replication as well as efficient expression of latent genes. Interestingly, Tip60 small molecule inhibitors, MG149 and NU9056, similarly inhibited latent and lytic genes, and reduced virion production in wild-type KSHV+/EBV- PEL, BCBL-1 cells. Long-term treatment with these Tip60 inhibitors selectively decreased the viability of KSHV-infected B lymphoma cells compared to uninfected cells. From this study, we conclude that Tip60 is important for KSHV infection and its associated cancer development, and Tip60 is therefore a potential target for future antiviral and anticancer therapeutics

Reactivation of Latent HIV-1 by Inhibition of BRD4

HIV-1 depends on many host factors for propagation. Other host factors, however, antagonize HIV-1 and may have profound effects on viral activation. Curing HIV-1 requires the reduction of latent viral reservoirs that remain in the face of antiretroviral therapy. Using orthologous genetic screens, we identified bromodomain containing 4 (BRD4) as a negative regulator of HIV-1 replication. Antagonism of BRD4, via RNA interference or with a small molecule inhibitor, JQ1, both increased proviral transcriptional elongation and alleviated HIV-1 latency in cell-line models. In multiple instances, JQ1, when used in combination with the NF-κB activators Prostratin or PHA, enhanced the in vitro reactivation of latent HIV-1 in primary T cells. These data are consistent with a model wherein BRD4 competes with the virus for HIV-1 dependency factors (HDFs) and suggests that combinatorial therapies that activate HDFs and antagonize HIV-1 competitive factors may be useful for curing HIV-1 infection

Data_Sheet_2_Inhibition of Tip60 Reduces Lytic and Latent Gene Expression of Kaposi’s Sarcoma-Associated Herpes Virus (KSHV) and Proliferation of KSHV-Infected Tumor Cells.docx

<p>Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus responsible for the development of Kaposi’s sarcoma, primary effusion lymphoma (PEL), and Multicentric Castleman’s disease in immunocompromised individuals. Despite the burden of these diseases there are few treatment options for afflicted individuals, due in part to our limited understanding of virus-host interactions. Tip60, a histone aceytltransferase (HAT) has been previously shown to interact with both the KSHV latency associated nuclear antigen protein (LANA), which is the main factor in maintaining the viral latent state, and ORF36, a viral kinase expressed in the lytic phase. We further investigated Tip60-virus interaction to ascertain Tip60’s role in the viral life cycle and its potential as a target for future therapeutics. Through modulation of Tip60 expression in HEK293T cells harboring a plasmid containing the KSHV viral episome, Bac36, we found that Tip60 is vital for both lytic replication as well as efficient expression of latent genes. Interestingly, Tip60 small molecule inhibitors, MG149 and NU9056, similarly inhibited latent and lytic genes, and reduced virion production in wild-type KSHV⁺/EBV^- PEL, BCBL-1 cells. Long-term treatment with these Tip60 inhibitors selectively decreased the viability of KSHV-infected B lymphoma cells compared to uninfected cells. From this study, we conclude that Tip60 is important for KSHV infection and its associated cancer development, and Tip60 is therefore a potential target for future antiviral and anticancer therapeutics.</p

Data_Sheet_1_Inhibition of Tip60 Reduces Lytic and Latent Gene Expression of Kaposi’s Sarcoma-Associated Herpes Virus (KSHV) and Proliferation of KSHV-Infected Tumor Cells.docx

<p>Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus responsible for the development of Kaposi’s sarcoma, primary effusion lymphoma (PEL), and Multicentric Castleman’s disease in immunocompromised individuals. Despite the burden of these diseases there are few treatment options for afflicted individuals, due in part to our limited understanding of virus-host interactions. Tip60, a histone aceytltransferase (HAT) has been previously shown to interact with both the KSHV latency associated nuclear antigen protein (LANA), which is the main factor in maintaining the viral latent state, and ORF36, a viral kinase expressed in the lytic phase. We further investigated Tip60-virus interaction to ascertain Tip60’s role in the viral life cycle and its potential as a target for future therapeutics. Through modulation of Tip60 expression in HEK293T cells harboring a plasmid containing the KSHV viral episome, Bac36, we found that Tip60 is vital for both lytic replication as well as efficient expression of latent genes. Interestingly, Tip60 small molecule inhibitors, MG149 and NU9056, similarly inhibited latent and lytic genes, and reduced virion production in wild-type KSHV⁺/EBV^- PEL, BCBL-1 cells. Long-term treatment with these Tip60 inhibitors selectively decreased the viability of KSHV-infected B lymphoma cells compared to uninfected cells. From this study, we conclude that Tip60 is important for KSHV infection and its associated cancer development, and Tip60 is therefore a potential target for future antiviral and anticancer therapeutics.</p

Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics

The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication

The CD225 Domain of IFITM3 is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication

IFITM3 is an interferon stimulated gene which inhibits the replication of multiple pathogenic viruses in vitro and in vivo. IFITM3 is a member of a large protein super family, whose members share a functionally-undefined area of high amino acid conservation, the CD225 domain. We performed mutational analyses of IFITM3 and identified multiple residues within the CD225 domain, consisting of the first intramembrane domain (IM1) and a conserved intracellular loop (CIL), that are required for restriction of both influenza A virus (IAV) and dengue virus (DENV) infection in vitro. Two phenylalanines within IM1 (F75 and F78) also mediate a physical association between IFITM proteins, and the loss of this interaction decreases IFITM3-mediated restriction. By extension, similar IM1-mediated associations may contribute to the functions of additional members of the CD225 domain family. IFITM3\u27s distal N-terminal domain is also needed for full anti-viral activity, including a tyrosine (Y20), whose alteration results in mislocalization of a portion of IFITM3 to the cell periphery and surface. Comparative analyses demonstrate that similar molecular determinants are needed for IFITM3\u27s restriction of both IAV and DENV. However, a portion of the CIL including Y99 and R87 is preferentially needed for inhibition of the orthomyxovirus. Several IFITM3 proteins engineered with rare single nucleotide polymorphisms demonstrated reduced expression or mislocalization, and these events were associated with enhanced viral replication in vitro, suggesting that possessing such alleles may impact an individual\u27s risk for viral infection. Based on this and other data, we propose a model for IFITM3-mediated restriction