A functional role for ADAM10 in human immunodeficiency virus type-1 replication.

BACKGROUND: Gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. Cells harboring a disrupted ADAM10 (A Disintegrin and Metalloprotease 10) allele survived reovirus infection, and subsequently ADAM10 was shown by RNA interference to be important for replication of HIV-1. RESULTS: Silencing ADAM10 expression with small interfering RNA (siRNA) 48 hours before infection significantly inhibited HIV-1 replication in primary human monocyte-derived macrophages and in CD4⁺ cell lines. In agreement, ADAM10 over-expression significantly increased HIV-1 replication. ADAM10 down-regulation did not inhibit viral reverse transcription, indicating that viral entry and uncoating are also independent of ADAM10 expression. Integration of HIV-1 cDNA was reduced in ADAM10 down-regulated cells; however, concomitant 2-LTR circle formation was not detected, suggesting that HIV-1 does not enter the nucleus. Further, ADAM10 silencing inhibited downstream reporter gene expression and viral protein translation. Interestingly, we found that while the metalloprotease domain of ADAM10 is not required for HIV-1 replication, ADAM15 and γ-secretase (which proteolytically release the extracellular and intracellular domains of ADAM10 from the plasma membrane, respectively) do support productive infection. CONCLUSIONS: We propose that ADAM10 facilitates replication at the level of nuclear trafficking. Collectively, our data support a model whereby ADAM10 is cleaved by ADAM15 and γ-secretase and that the ADAM10 intracellular domain directly facilitates HIV-1 nuclear trafficking. Thus, ADAM10 represents a novel cellular target class for development of antiretroviral drugs.

Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain.

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4(+) T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry.

Inhibition of influenza A virus replication by antagonism of a PI3K-AKT-mTOR pathway member identified by gene-trap insertional mutagenesis.

BACKGROUND: Host genes serving potential roles in virus replication may be exploited as novel antiviral targets. METHODS: Small interfering RNA (siRNA)-mediated knockdown of host gene expression was used to validate candidate genes in screens against six unrelated viruses, most importantly influenza. A mouse model of influenza A virus infection was used to evaluate the efficacy of a candidate FDA-approved drug identified in the screening effort. RESULTS: Several genes in the PI3K-AKT-mTOR pathway were found to support broad-spectrum viral replication in vitro by RNA interference. This led to the discovery that everolimus, an mTOR inhibitor, showed in vitro antiviral activity against cowpox, dengue type 2, influenza A, rhino- and respiratory syncytial viruses. In a lethal mouse infection model of influenza A (H1N1 and H5N1) virus infection, everolimus treatment (1 mg/kg/day) significantly delayed death but could not prevent mortality. Fourteen days of treatment was more beneficial in delaying the time to death than treatment for seven days. Pathological findings in everolimus-treated mice showed reduced lung haemorrhage and lung weights in response to infection. CONCLUSIONS: These results provide proof of concept that cellular targets can be identified by gene knockout methods, and highlight the importance of the PI3K-AKT-mTOR pathway in supporting viral infections.

Identification of cellular proteins required for replication of human immunodeficiency virus type 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 ß-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.

A critical role for CD63 in HIV replication and infection of macrophages and cell lines.

HIV infection typically involves interaction of Env with CD4 and a chemokine coreceptor, either CCR5 or CXCR4. Other cellular factors supporting HIV replication have also been characterized. We previously demonstrated a role for CD63 in early HIV infection events in macrophages via inhibition by anti-CD63 antibody pretreatment. To confirm the requirement for CD63 in HIV replication, we decreased CD63 expression using CD63-specific short interfering RNAs (siRNA), and showed inhibition of HIV replication in macrophages. Surprisingly, pretreatment with CD63 siRNA not only silenced CD63 expression by 90%, but also inhibited HIV-1 replication in a cultured cell line (U373-MAGI) which had been previously shown to be insensitive to CD63 monoclonal antibody inhibition. Although the anti-CD63 antibody was previously shown to inhibit early HIV infection events only in macrophages, we now show a potential role for CD63 in later HIV replication events in macrophages and cell lines. Further delineation of the role of CD63 in HIV replication may lead to development of novel therapeutic compounds.

Rab 5 is required for the cellular entry of dengue and West Nile viruses.

The mechanisms of cellular entry of dengue and West Nile viruses are not well characterized. We show that both these viruses enter HeLa cells by clathrin-dependent endocytosis and require vacuolar acidic pH. Inhibition of the GTPase Rab 5 or 7, which regulates transport to early or late endosomes, respectively, demonstrated that Rab 5 was essential for survival of both dengue and West Nile virus. These data broaden our understanding of the pathways required for productive dengue and West Nile virus infection and may facilitate new strategies for combating disease.

Internalized Pseudomonas exotoxin A can exploit multiple pathways to reach the endoplasmic reticulum.

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the intoxication of mammalian cells by Pseudomonas exotoxin A (PEx). The toxin binds the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Here, we show that in HeLa cells, PEx recruits a proportion of this receptor to detergent-resistant microdomains (DRMs). Uptake of receptor-bound PEx involves transport steps both directly from early endosomes to the trans-Golgi network (TGN) independently of Rab9 function and from late endosomes to the TGN in a Rab9-dependent manner. Furthermore, treatments that simultaneously perturb both Arf1-dependent and Rab6-dependent retrograde pathways show that PEx can use multiple routes to reach the ER. The Rab6-dependent route has only been described previously for cargo with lipid-sorting signals. These findings suggest that partial localization of PEx within DRM permits a choice of trafficking routes consistent with a model that DRM-associated toxins reach the ER on a lipid-dependent sorting pathway whilst non-DRM-associated PEx exploits the previously characterized KDEL receptor-mediated uptake pathway. Thus, unexpectedly, an ER-directed toxin with a proteinaceous receptor shows promiscuity in its intracellular trafficking pathways, exploiting routes controlled by both lipid- and protein-sorting signals.

Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus.

Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. By using gene trap insertional mutagenesis, we identified Rab9, which mediates late-endosome-to-trans-Golgi-network trafficking, among several candidate host genes whose disruption allowed the survival of Marburg virus-infected cells, suggesting that Rab9 is utilized in Marburg replication. Although Rab9 has not been implicated in human immunodeficiency virus (HIV) replication, previous reports suggested that the late endosome is an initiation site for HIV assembly and that TIP47-dependent trafficking out of the late endosome to the trans-Golgi network facilitates the sorting of HIV Env into virions budding at the plasma membrane. We examined the role of Rab9 in the life cycles of HIV and several unrelated viruses, using small interfering RNA (siRNA) to silence Rab9 expression before viral infection. Silencing Rab9 expression dramatically inhibited HIV replication, as did silencing the host genes encoding TIP47, p40, and PIKfyve, which also facilitate late-endosome-to-trans-Golgi vesicular transport. In addition, silencing studies revealed that HIV replication was dependent on the expression of Rab11A, which mediates trans-Golgi-to-plasma-membrane transport, and that increased HIV Gag was sequestered in a CD63+ endocytic compartment in a cell line stably expressing Rab9 siRNA. Replication of the enveloped Ebola, Marburg, and measles viruses was inhibited with Rab9 siRNA, although the non-enveloped reovirus was insensitive to Rab9 silencing. These results suggest that Rab9 is an important cellular target for inhibiting diverse viruses and help to define a late-endosome-to-plasma-membrane vesicular transport pathway important in viral assembly.

High resolution crystal structure of human Rab9 GTPase: a novel antiviral drug target.

Rab GTPases and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome to trans-Golgi transport and has recently been found to be a key cellular component for human immunodeficiency virus-1, Ebola, Marburg, and measles virus replication, suggesting that it may be a novel target in the development of broad spectrum antiviral drugs. As part of our structure-based drug design program, we have determined the crystal structure of a C-terminally truncated human Rab9 (residues 1-177) to 1.25-A resolution. The overall structure shows a characteristic nucleotide binding fold consisting of a six-stranded beta-sheet surrounded by five alpha-helices with a tightly bound GDP molecule in the active site. Structure-based sequence alignment of Rab9 with other Rab proteins reveals that its active site consists of residues highly conserved in the Rab GTPase family, implying a common catalytic mechanism. However, Rab9 contains seven regions that are significantly different in conformation from other Rab proteins. Some of those regions coincide with putative effector-binding sites and switch I and switch II regions identified by structure/sequence alignments. The Rab9 structure at near atomic resolution provides an excellent model for structure-based antiviral drug design.

Discordance at human leukocyte antigen-DRB3 and protection from human immunodeficiency virus type 1 transmission.

Host human leukocyte antigens (HLAs) integrated into the human immunodeficiency virus (HIV) type 1 envelope could theoretically determine, as in tissue transplants, whether HIV-1 is "rejected" by exposed susceptible persons, preventing transmission. HLA discordance (mismatch) was examined among 45 heterosexual partner pairs in which at least 1 partner was HIV-1 infected and exposure or transmission between partners had occurred. Immunologic discordance at class II HLA-DRB3 (present in the HIV donor partner but absent in the recipient partner) was associated with lack of transmission of HIV-1. Eight (35%) of 23 partner pairs in which HIV-1 transmission did not occur were immunologically discordant at HLA-DRB3, compared with 0 of 11 partner pairs in which HIV-1 transmission did occur (P=.027). Further investigation of the roles of class II HLAs in HIV-1 transmission and as possible components of HIV-1 vaccines should be pursued.

Broad human immunodeficiency virus (HIV)-specific T cell responses to conserved HIV proteins in HIV-seronegative women highly exposed to a single HIV-infected partner.

Eighteen highly exposed but persistently seronegative (HEPS) women (HW) and their human immunodeficiency virus (HIV) type 1-seropositive male partners were studied for HIV-specific T cells and other host factors. Circulating HIV-specific T cells were measured by interferon-gamma enzyme-linked immunospot assays, using recombinant vaccinia virus vectors expressing HIV proteins. Nine (50%) of the HW and all HIV-seropositive persons had HIV-specific T cell responses. Only 2 (22%) of the HEPS responders recognized Env, compared with 94% of HIV-seropositive persons. A high percentage (75%) of the HW with HIV-specific T cell responses reported recent HIV exposure. Remarkably, however, long-lived HIV-specific T cells were detected in 2 HW who had an extended period (>3.9 years) of no HIV exposure. These findings have important implications for HIV vaccine design.

Frequent human leukocyte antigen class I alleles are associated with higher viral load among HIV type 1 seroconverters in Thailand.

The loss of viral control by the host may be due to the evolution of viruses with mutations that limit presentation by human leukocyte antigen (HLA) to cytotoxic T cells. The authors hypothesized that the consequence of such evolution might be that persons with common HLA class I alleles would be less able to control viremia, on average, than would those with rare alleles. HLA class I typing was completed for 128 injection drug users who seroconverted in a prospective cohort study in Bangkok, Thailand. Logistic regression was used to model viral load (greater than or equal to the median) at 9 and 12 months after seroconversion with an HLA score that profiled the relative prevalence of each individual's alleles. At 12 months after seroconversion, injection drug users with the most common HLA alleles (highest quartile HLA score) had an almost 4-fold increased risk for higher viral load (> or = 32,055 copies/mL) than injection drug users with less common HLA alleles (adjusted odds ratio, 3.92; 95% confidence interval, 1.3-11.8). These findings support the importance of frequency-dependent effects of host genes on HIV type 1 evolution in different populations and suggest that HLA-driven viral evolution critically influences control of viremia in early HIV type 1 infection.