Thomas W. Hodge

Rab 5 Is Required for the Cellular Entry of Dengue and West Nile Viruses

ABSTRACT 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.

Rab9 GTPase Is Required for Replication of Human Immunodeficiency Virus Type 1, Filoviruses, and Measles Virus

ABSTRACT 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 nonenveloped 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.

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 α2‐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.

An HLA-Directed Molecular and Bioinformatics Approach Identifies New HLA-A11 HIV-1 Subtype E Cytotoxic T Lymphocyte Epitopes in HIV-1-Infected Thais

Only limited cytotoxic T lymphocyte (CTL) epitope mapping has been done in nonsubtype B HIV-infected persons. We used molecular immunogenetic tools to determine HIV-specific CTL responses in HIV-1 Env subtype E-infected female sex workers (FSWs) from northern Thailand, where more than 50% of the population is HLA-A11 positive. EpiMatrix, a computer-based T cell epitope prediction algorithm, and a manual editing approach were used to predict 77 possible HLA-A11 CTL epitopes in HIV-1, some of which were conserved between subtypes B and E. MHC binding of these peptides was determined in an HLA-A11 stabilization assay, and binding peptides were tested for CTL recognition in eight HLA-A11-positive FSWs. Subtype E versions of known HLA-A2 subtype B HIV epitopes were also tested in four HLA-A2 positive FSWs. CTL responses were detected in all HLA-A11-positive and in three of four HLA-A2-positive persons. Among the 12 FSWs responses to peptides were found to Pol in 9 (75%), Env in 7 (58%), Nef in 5 (42%), and Gag in 5 (42%), and to conserved epitopes in 8 (67%). To identify HLA-A11 CTL epitopes in the absence of prediction tools, it would have been necessary to test almost 3000 10-mer peptides. EpiMatrix and manual predictions reduced this number to 77, of which 26 were MHC binding and 12 were CTL epitopes. Six of these HLA-A11 CTL epitopes have not been previously reported and are located in RT, gp120, and gp41. This report of CTL responses in subtype E-infected individuals defines epitopes that may be useful in HIV pathogenesis or vaccine studies.

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.

Production of a Novel Viral Suppressive Activity Associated with Resistance to Infection among Female Sex Workers Exposed to HIV Type 1

To investigate mechanisms of natural resistance to human immunodeficiency virus type 1 (HIV-1), we obtained blood samples from eight women who remained HIV-1 negative after > 3 years of high-risk sex work in Chiang Rai, Thailand. CD4+ T lymphocytes from these highly exposed, persistently seronegative (HEPS) women were readily infectable in vitro with HIV-1 subtypes B and E. Autologous CD8+ cell suppression of both HIV-1 subtypes was evident in HEPS infection cultures, but to an extent also observed in cultures from non-HIV-exposed individuals. Furthermore, production of beta-chemokines was not enhanced in HEPS cultures. However, HEPS cultures displayed significantly enhanced production of a soluble activity that suppressed postintegrated HIV-1 replication. This activity was the unique product of CD4+ T cell and monocyte cocultures. Therefore, although HEPS individuals are apparently susceptible to infection, the production of a postintegrated HIV-1 suppressive activity during monocyte-T cell interactions might protect against the establishment of infection by limiting viral dissemination.

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.

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.

Polymorphisms in the CCR5 Coding and Noncoding Regions among HIV Type 1-Exposed, Persistently Seronegative Female Sex-Workers from Thailand

Resistance to HIV-1 infection despite repeated exposures has been associated with one or more HIV-specific responses, enhanced nonspecific immune modifications, and/or host genetic polymorphisms in certain individuals (highly exposed, persistently seronegative, HEPS). In the present investigation, we focused on the CCR5 gene polymorphisms and the association of such mutations to resistance to HIV-1 infection among 12 HEPS women in Chiang Rai, northern Thailand, and compared our findings with data from 10 HIV-1-infected and 9 HIV-1-uninfected unexposed women from the same geographic area. Although we have previously shown that none of the Thai women carried the Delta32 mutation, further analysis of the CCR5 coding gene region revealed that none of the women had other mutations that affect coreceptor activity (C101X or FS299) or chemokine responses (C20S, A29S, L55Q, C178R). Analysis of the CCR5 promoter region revealed that the CCR5 haplogroup C (HHC; 60%) was the predominant haplogroup among these women. Comparative analysis of the frequencies of different haplogroups among the three groups did not reveal any statistically significant differences (p > 0.05). However, we did find that two individuals from the HEPS group were homozygous for HHF*2 (the CCR2b- 64I bearing haplogroup) compared to none from the HIV-1-infected and -uninfected groups. There was no detectable difference in specific CCR5 haplogroups and their ability to mediate env fusion or to mediate HIV-1 infection in vitro. These data suggest that homozygosity of the HHF*2 haplogroup may be one of the factors that mediate resistance to HIV-1 infection in this cohort of HEPS women.

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 (≥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.