Question

How is each of these proteins expressed in HIV infected cells, and how does each function in the HIV replication cycle?

The three proteins in question are vpu, vpx, and tat.

Answer 1

Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.

HIV-1 is an obligate intracellular pathogen encoding only 15 proteins,1 and is thus dependent upon host factors for productive infection.2 HIV-1 uses CD4 as its primary receptor and the major coreceptors CCR5 and CXCR4 to gain entrance into host cells.3–5 Following entry, the viral core is partially disassembled, allowing viral RNA and proteins access to the cytoplasm. The viral reverse transcriptase (RT) converts viral RNA into DNA, which is then transported into the nucleus via the preintegration complex and integrated into the host genome. Subsequently, viral mRNAs are expressed, transported to the cytoplasm, and translated as precursor to viral assembly and budding. HIV-1 depends on host factors/pathways for successful completion of virtually every step in the viral life cycle.6,7 Recent large-scale siRNA screens have revealed hundreds of candidate host genes that potentially augment HIV-1 replication, illuminating new cellular pathways involved in the viral life cycle.8–16

The majority of cellular targets implicated in HIV-1 replication have been identified in genomic siRNA screens. Due to the large numbers of genes tested, most genomic siRNA screens have employed reporter systems, replicon systems, or pseudotyped viruses that identify cellular factors important for a limited subset of stages in the viral life cycle. The predesigned siRNA libraries used for global screens have not included siRNAs targeting many unknown/hypothetical genes or nonprotein coding RNAs. We have used gene-trap insertional mutagenesis17 as a complementary high-throughput screening (HTS) forward genetics approach to discover mammalian genes mediating infection, by identifying genes whose disruption confers resistance to otherwise lytic viruses.18–21 Using this technology, cellular genes are disrupted (trapped) with a recombinant Maloney murine leukemia virus (MMLV) virus encoding a promoterless neomycin resistance gene that randomly integrates into the chromosome. Integration of the MMLV vector between cellular promoters and early exons allows neomycin selection and derivation of gene-trap libraries with subverted gene expression. Infecting gene-trap libraries with a lytic virus typically results in >99.99% cytopathic effects (CPE), allowing high stringency selection of clonal cell lines surviving infection. Trapped genes in resistant clones are identified by sequencing across MMLV vector/genomic DNA junction sites. Importantly, gene-trap studies can identify genes important for any step in the replication cycle, do not require prior knowledge of the gene, and the roles of a prioritized subset of all human genes in viral infection can be confirmed independently by RNA interference (RNAi).

Lytic viral selection has been employed previously to identify HIV-1-dependency factors in Jurkat T cells whose silencing conferred survival in a genome-wide shRNA screen.14 In our experimental system using HeLa cell-derived gene-trap libraries, the maximum CPE observed following HIV-1 infection was ∼90% (MOI=100), which was an unacceptably high background of surviving cells to isolate resistant clones. However, as unrelated lytic viruses show conserved utilization of cellular genes,19,20 we tested 87 disrupted genes identified in cowpox, Ebola, influenza A, Marburg, and reovirus gene-trapping studies for conserved roles in HIV-1 replication. An additional seven targets were secondarily selected for investigation in light of their known pathway associations with critical trapped targets. In siRNA validation screens in an immortalized cell line, we identified several novel host genes, which support HIV-1 replication and other viruses. The roles of critical genes in HIV-1 replication were confirmed in primary human macrophages, and evidence is presented that most of these proteins may facilitate replicative steps prior to or including transactivator of transcription (Tat)-mediated gene transcription. Bioinformatics analyses suggested that many of the gene products participate in conserved pathways or functions important for HIV-1 replication, including gene transcription, signal transduction, mRNA splicing, protein ubiquitination, vesicular transport, and autophagy.

Cell culture

Primary macrophages were purified from fresh peripheral blood mononuclear cells by adherence to plastic tissue culture dishes, as described.22 Using this method, we previously showed purity of isolated mononuclear phagocytes of >95% by flow cytometry.23 Briefly, primary monocyte-derived macrophages were isolated from healthy HIV-1-negative blood donors by Ficoll-Hypaque centrifugation followed by adherence for 7 days to plastic Petri dishes. During differentiation, macrophages were cultured in Iscove's modified Dulbecco's medium supplemented with 20% fetal calf serum and 10% of human AB serum.

The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme Inc. U373-MAGI-CCR5 cells, obtained through the NIH AIDS Research and Reference Reagent Program (contributed by Dr. Michael Emerman and Dr. Adam Geballe), are a cell line derived from a glioblastoma that has been modified by stable transfection of expression vectors encoding LTR-β-galactosidase and CCR5 to enable infection by HIV-1.24

Gene-trap library construction

The U3neoSV1 retrovirus shuttle vector25 was obtained from H. Earl Ruley (Vanderbilt University). RIE-1, MDCK, and Vero E6 cells served as suitable parental cell lines for the preparation of gene-trap libraries, as they are efficiently killed by infection with reovirus (RIE-1 cells), influenza A (MDCK cells), and either cowpox, Ebola, or Marburg viruses (Vero E6 cells). RIE-1, MDCK, and Vero E6 gene-trap libraries were prepared as described.19–21

Generation of cell lines resistant to lytic viral infection from gene-trap libraries

Clonal reovirus, Ebola, and Marburg virus-resistant cell lines were derived from parental RIE-1 (reovirus, Lang strain) and Vero E6 (Ebola, 1976 Mayinga strain and Marburg, 1967 Voege strain) gene-trap library cells as described.19–21 Vero E6 library cells were also used to select cell lines surviving cowpox infection (Brighton strain), and MDCK library cells were used for influenza (A/PR/8/34 strain) studies. Briefly, gene-trap libraries, each harboring approximately 104 gene entrapment events, were expanded to 80–90% confluency until approximately 103 daughter cells represented each clone. Vero E6 cells were infected with cowpox using a multiplicity of infection (MOI) of 0.01, and MDCK cells were infected with influenza A using an MOI of 0.1. Infection proceeded at 37°C until >90% of cells were dead, at which point the medium was changed every 2–3 days to remove dead cells. Dead cells were removed by aspiration for 2–3 weeks until surviving clones were visually observed. Individual clones were detached with trypsin and isolated into separate wells of 24-well plates. Following expansion, resistance confirmation was performed by replica plating clones into duplicate 24-well plates, and reinfecting clonal cell lines with a 20-fold higher MOI than used in the initial viral selection for 2 h at 37°C before changing the medium. Clones that showed >70% survival following reinfection were selected for expansion to identify trapped genes.

U3neoSV1 shuttle vector rescue and sequencing

Genomic DNAs from clonal virus-resistant cell lines were extracted using a QIAamp DNA Blood Mini kit (QIAGEN, Inc., Valencia, CA). Shuttle vectors and genomic DNA flanking the U3neoSV1 integration site were recovered by restriction enzyme digests of genomic DNA, self-ligation, transformation into Escherichia coli, and sequencing the resultant carbenicillin-resistant plasmids to identify trapped genes, as described.19–21

Viruses and antibodies

The following HIV-1 strains were purchased from the Virology Core Facility, Center for AIDS Research at Baylor College of Medicine, Houston, TX: M-tropic HIV-1SF162 and HIV-1ADA, dual-tropic HIV-189.6, which is an HIV-1 laboratory adapted strain originally isolated from infected individuals. HIV-1SX is a chimeric M-tropic virus encoding the majority of the HIV-1JRFL envelope protein in an HIV-1NL4-3 backbone. Antibodies for detecting RAB9A, CCR5, DHX8, KALRN, UBA3, and HAP1 protein expression in U373 cell lines and macrophages were obtained from Abcam Inc. An anti-RAB11A antibody was obtained from Invitrogen. Antibodies recognizing CD4, ERBB2IP, VMP1, and UBE2E3 were obtained from Abnova. An anti-β-actin antibody was obtained from Santa Cruz Biotechnology. These antibodies were used to perform Western dot-blot analysis.

Western dot-blot analysis

Cellular expression and downregulation of cellular proteins were analyzed in Western dot-blot analysis using primary antibodies specific for cellular proteins and secondary antihuman antibody conjugated to HRP (obtained from Santa Cruz Biotechnology).

siRNA transfections

siRNA SMARTpools were obtained from Dharmacon. TZM-bl cells were transfected with siRNAs as described.19 Primary human macrophages were transfected with siRNAs using oligofectamine (Invitrogen) according to the manufacturer's instructions. Primary macrophages adherent for 5 days were seeded overnight in 96-well plate format (1×105 cells / well) and subsequently transfected with 50 nM siRNA in serum-free Iscove's medium. Cells were fed with Iscove's medium and 20% fetal calf serum (FCS) after 4 h to terminate transfection. To measure protein expression following RNAi, cells were lysed at 48 h posttransfection in phosphate-buffered saline (PBS) with 1% Triton X-100 in preparation for Western dot-blot analysis. U373-MAGI-CCR5 cells were transfected with 50 nM siRNA 24 h after plating at 1×104 cells per well in 96-well microtiter plates. Protein expression and downregulation in U373-MAGI-CCR5 cells were assessed at 48 h post-siRNA transfection by lysing cells in 1% Triton X-100 in PBS and detecting target proteins by Western dot-blot analysis.

Real-time semiquantitative PCR

Total mRNA was isolated from parental and siRNA-transfected cells using the RNeasy Kit (Qiagen, Inc.), and reverse transcribed using random hexamers (Applied Biosystems). Real time PCR was performed as described26 using an Mx4000 Multiplex Quantitative PCR System (Stratagene). Real time PCR detection assays for all siRNA target genes described in this study were from Applied Biosystems. Target gene mRNA expression levels were normalized to hypoxanthine-guanine phosphoribosyltransferase (HGPRT) mRNA expression levels, using the human HGPRT TaqMan assay kit (Applied Biosystems).

HIV-1 p24 and β-galactosidase assays

HIV-1 p24 ELISA assays were used to assess HIV-1 production in TZM-bl cells and primary human macrophages, and β-galactosidase assays measuring HIV-1 Tat-dependent gene expression were performed in U373-MAGI-CCR5 cells. HIV-1 p24 assays were performed in TZM-bl cells as described previously.19 Primary macrophages adherent for 7 days were seeded overnight in 96-well plates (1×105 cells per well) and transfected after 24 h with 50 nM siRNAs for 48 h. Subsequently, macrophages were infected for 2 h with either SF162, ADA, or 89.6 HIV-1 strains (MOI=0.02), after which the medium was changed to remove the inoculum. Cells were fed with fresh medium on day 4 postinoculation, and supernatants were assessed for virus p24 production using the Antigen Capture ELISA assay (ImmunoDiagnostics) on day 7 postinoculation.

U373-MAGI-CCR5 cells express β-galactosidase under the control of the HIV-1 LTR, which is transactivated by the HIV-1 Tat protein. U373-MAGI-CCR5 cells (1×104/well) were plated in 96-well microtiter plates 12 h before siRNA transfections. At 48 h posttransfection the cells were infected with HIV-1 strains (MOI=0.02). After 2 h postinoculation at 37°C, the medium was changed and β-galactosidase activity was measured 48 h after infection using the Beta-Glo Assay System (Promega).

Cytotoxicity assays

Cytotoxicity of siRNA was measured using the aCella-Tox Bioluminescence Cytotoxicity assay kit according to the protocol provided by the manufacturer (Cell Technology Inc., Mountain View, CA). This assay detects secreted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells with compromised membrane integrity. Cell supernatants were harvested at 48 h posttransfection and GAPDH enzyme activity was measured by luminometry.

Bioinformatics analyses

Eight genes implicated in HIV-1 replication during siRNA screens (DHX8, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1) were analyzed using Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Inc.). To allow comparison with prior genome-wide siRNA screens by Brass et al.,16 Konig et al.,11 Zhou et al.,15 Yeung et al.,14 and Liu et al.,27 as well as a systematic affinity tagging mass spectrometry study by Jäger et al.,28 additional IPA analyses were performed on their 281, 292, 232, 252, 114, and 497 gene datasets, respectively. These analyses identified canonical pathways from the IPA library of canonical pathways that were statistically significant compared to the datasets from each study, based on Fisher's exact test. Canonical pathways with a calculated p-value less than 0.01 were considered significant, implying that the probability of a chance association between genes in the dataset and the canonical pathway is less than 1%. Functional analyses were also performed with the above datasets, identifying biological functions that were statistically significant with functions in the Ingenuity Knowledge Base based on the right-tailed Fisher's exact test (p-value<0.05).