Disparate outcomes in Hispanic patients with metabolic dysfunction-associated steatotic liver disease/steatohepatitis and type 2 diabetes: Large cohort study.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) are a growing health burden across a significant portion of the global patient population. However, these conditions seem to have disparate rates and outcomes between different ethnic populations. The combination of MASLD/MASH and type 2 diabetes increases the risk of hepatocellular carcinoma (HCC), and Hispanic patients experience the greatest burden, particularly those in South Texas. AIM: To compare outcomes between Hispanic and non-Hispanic patients in the United States, while further focusing on the Hispanic population within Southeast Texas to determine whether the documented disparity in outcomes is a function of geographical circumstance or if there is a more widespread reason that all clinicians must account for in prognostic consideration. METHODS: This cohort analysis was conducted with data obtained from TriNetX, LLC ("TriNetX"), a global federated health research network that provides access to deidentified medical records from healthcare organizations worldwide. Two cohort networks were used: University of Texas Medical Branch (UTMB) hospital and the United States national database collective to determine whether disparities were related to geographic regions, like Southeast Texas. RESULTS: This study findings revealed Hispanics/Latinos have a statistically significant higher occurrence of HCC, type 2 diabetes mellitus, and liver fibrosis/cirrhosis in both the United States and the UTMB Hispanic/Latino groups. All-cause mortality in Hispanics/Latinos was lower within the United States group and not statistically elevated in the UTMB cohort. CONCLUSION: This would appear to support that Hispanic patients in Southeast Texas are not uniquely affected compared to the national Hispanic population.

Coronavirus Disease 2019, Diabetes, and Inflammation: A Systemic Review.

People with cardiometabolic diseases [namely type 2 diabetes (T2D), obesity, or metabolic syndrome] are more susceptible to coronavirus disease 2019 (COVID-19) infection and endure more severe illness and poorer outcomes. Hyperinflammation has been suggested as a common pathway for both diseases. To examine the role of inflammatory biomarkers shared between COVID-19 and cardiometabolic diseases, we reviewed and evaluated published data using PubMed, SCOPUS, and World Health Organization COVID-19 databases for English articles from December 2019 to February 2022. Of 248 identified articles, 50 were selected and included. We found that people with diabetes or obesity have (i) increased risk of COVID-19 infection; (ii) increased risk of hospitalization (those with diabetes have a higher risk of intensive care unit admissions) and death; and (iii) heightened inflammatory and stress responses (hyperinflammation) to COVID-19, which worsen their prognosis. In addition, COVID-19-infected patients have a higher risk of developing T2D, especially if they have other comorbidities. Treatments controlling blood glucose levels and or ameliorating the inflammatory response may be valuable for improving clinical outcomes in these patient populations. In conclusion, it is critical for health care providers to clinically evaluate hyperinflammatory states to drive clinical decisions for COVID-19 patients.

Multispectral Imaging Enables Characterization of Intrahepatic Macrophages in Patients With Chronic Liver Disease.

Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared with stellate cells, the role of macrophages in the development of fibrosis remains unclear. Multispectral imaging allows detection of multiple markers in situ in human formalin-fixed, paraffin-embedded tissue. This cutting-edge technology is ideal for analyzing human liver tissues, as it allows spectral unmixing of fluorophore signals, subtraction of auto-fluorescence, and preservation of hepatic architecture. We analyzed five different antibodies commonly observed on macrophage populations (CD68, MAC387, CD163, CD14, and CD16). After optimization of the monoplex stains and development of a Spectral Library, we combined all of the antibodies into a multiplex protocol and used them to stain biopsies collected from representative patients with chronic liver diseases, including chronic hepatitis C, nonalcoholic steatohepatitis, and autoimmune hepatitis. Various imaging modalities were tested, including cell phenotyping, tissue segmentation, t-distributed stochastic neighbor embedding plots, and phenotype matrices that facilitated comparison and visualization of the identified macrophage and other cellular profiles. We then tested the feasibility of this platform to analyze numerous regions of interest from liver biopsies with multiple patients per group, using batch analysis algorithms. Five populations showed significant differences between patients positive for hepatitis C virus with advanced fibrosis when compared with controls. Three of these were significantly increased in patients with advanced fibrosis when compared to controls, and these included CD163+CD16+, CD68+, and CD68+MAC387+. Conclusion: Spectral imaging microscopy is a powerful tool that enables in situ analysis of macrophages and other cells in human liver biopsies and may lead to more personalized therapeutic approaches in the future.

The Medical Student Summer Research Program at the University of Texas Medical Branch at Galveston: building research foundations.

BACKGROUND: Interest in incorporating research into the medical school curriculum has grown over the years. One of the challenges involved with providing research to medical students is developing programs that allow a large number of students to perform research. This involves securing faculty to mentor students in the design of research projects. In order to accommodate students with research interests, well-established research programs must be implemented. OBJECTIVE: This article describes the design and implementation of a curriculum-based research program for medical students at the University of Texas Medical Branch (UTMB) at Galveston. The main objective of this article is to describe the program for the purpose of assisting other medical schools to develop a similar student research program. DESIGN: At UTMB we established a Medical Student Summer Research Program (MSSRP) that occurred between the first year and the second year of medical school. Between the years 2000-2017, MSSRP accommodated a minimum of 39 and a maximum of 90 students during an 8 week period. Two surveys were conducted to collect students' views on how MSSRP affected their interest in research. We performed a proportion statistical analysis on the data from both surveys in order to determine the significance of the responses. RESULTS: The benefit of MSSRP is that it provided medical students with an exposure to research. According to the proportions test, the responses were statistically significant with 85% of 26 third and fourth year students stating they would continue to incorporate research into their medical careers; 75% stating that MSSRP increased their interest in research; and 85% responding that MSSRP helped them to understand research methodology. CONCLUSIONS: MSSRP is a curriculum-based program that provides a framework to other medical institutions interested in the development of similar student research programs and provides students the exposure and option to continue with research as a component of their medical profession.

Fibrogenic Gene Expression in Hepatic Stellate Cells Induced by HCV and HIV Replication in a Three Cell Co-Culture Model System.

Retrospective studies indicate that co-infection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) accelerates hepatic fibrosis progression. We have developed a co-culture system (MLH) comprising primary macrophages, hepatic stellate cells (HSC, LX-2), and hepatocytes (Huh-7), permissive for active replication of HCV and HIV, and assessed the effect of these viral infections on the phenotypic changes and fibrogenic gene expression in LX-2 cells. We detected distinct morphological changes in LX-2 cells within 24 hr post-infection with HCV, HIV or HCV/HIV in MLH co-cultures, with migration enhancement phenotypes. Human fibrosis microarrays conducted using LX-2 cell RNA derived from MLH co-culture conditions, with or without HCV and HIV infection, revealed novel insights regarding the roles of these viral infections on fibrogenic gene expression in LX-2 cells. We found that HIV mono-infection in MLH co-culture had no impact on fibrogenic gene expression in LX-2 cells. HCV infection of MLH co-culture resulted in upregulation (>1.9x) of five fibrogenic genes including CCL2, IL1A, IL1B, IL13RA2 and MMP1. These genes were upregulated by HCV/HIV co-infection but in a greater magnitude. Conclusion: Our results indicate that HIV-infected macrophages accelerate hepatic fibrosis during HCV/HIV co-infection by amplifying the expression of HCV-dependent fibrogenic genes in HSC.

Sequential Dysfunction and Progressive Depletion of Candida albicans-Specific CD4 T Cell Response in HIV-1 Infection.

Loss of immune control over opportunistic infections can occur at different stages of HIV-1 (HIV) disease, among which mucosal candidiasis caused by the fungal pathogen Candida albicans (C. albicans) is one of the early and common manifestations in HIV-infected human subjects. The underlying immunological basis is not well defined. We have previously shown that compared to cytomegalovirus (CMV)-specific CD4 cells, C. albicans-specific CD4 T cells are highly permissive to HIV in vitro. Here, based on an antiretroviral treatment (ART) naïve HIV infection cohort (RV21), we investigated longitudinally the impact of HIV on C. albicans- and CMV-specific CD4 T-cell immunity in vivo. We found a sequential dysfunction and preferential depletion for C. albicans-specific CD4 T cell response during progressive HIV infection. Compared to Th1 (IFN-γ, MIP-1ß) functional subsets, the Th17 functional subsets (IL-17, IL-22) of C. albicans-specific CD4 T cells were more permissive to HIV in vitro and impaired earlier in HIV-infected subjects. Infection history analysis showed that C. albicans-specific CD4 T cells were more susceptible to HIV in vivo, harboring modestly but significantly higher levels of HIV DNA, than CMV-specific CD4 T cells. Longitudinal analysis of HIV-infected individuals with ongoing CD4 depletion demonstrated that C. albicans-specific CD4 T-cell response was preferentially and progressively depleted. Taken together, these data suggest a potential mechanism for earlier loss of immune control over mucosal candidiasis in HIV-infected patients and provide new insights into pathogen-specific immune failure in AIDS pathogenesis.

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.

Gp120 in the pathogenesis of human immunodeficiency virus-associated pain.

OBJECTIVE: Chronic pain is a common neurological comorbidity of human immunodeficiency virus (HIV)-1 infection, but the etiological cause remains elusive. The objective of this study was to identify the HIV-1 causal factor that critically contributes to the pathogenesis of HIV-associated pain. METHODS: We first compared the levels of HIV-1 proteins in postmortem tissues of the spinal cord dorsal horn (SDH) from HIV-1/acquired immunodeficiency syndrome patients who developed chronic pain (pain-positive HIV-1 patients) and HIV-1 patients who did not develop chronic pain (pain-negative HIV-1 patients). Then we used the HIV-1 protein that was specifically increased in the pain-positive patients to generate mouse models. Finally, we performed comparative analyses on the pathological changes in the models and the HIV-1 patients. RESULTS: We found that HIV-1 gp120 was significantly higher in pain-positive HIV-1 patients (vs pain-negative HIV-1 patients). This finding suggested that gp120 was a potential causal factor of the HIV-associated pain. To test this hypothesis, we used a mouse model generated by intrathecal injection of gp120 and compared the pathologies of the model and the pain-positive human HIV-1 patients. The results showed that the mouse model and pain-positive human HIV-1 patients developed extensive similarities in their pathological phenotypes, including pain behaviors, peripheral neuropathy, glial reactivation, synapse degeneration, and aberrant activation of pain-related signaling pathways in the SDH. INTERPRETATION: Our findings suggest that gp120 may critically contribute to the pathogenesis of HIV-associated pain.

Cultivated vaginal microbiomes alter HIV-1 infection and antiretroviral efficacy in colonized epithelial multilayer cultures.

There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.

The dual role of tetraspanin CD63 in HIV-1 replication.

BACKGROUND: Previously, we showed that the tetraspanin membrane protein CD63 mediates both early and post-integration stages of the HIV-1 replication cycle. The temporal roles of CD63 were discerned using monoclonal antibodies and small interfering RNAs (siRNAs) to block CD63 function, and determining which of the sequential steps in HIV-1 replication were disrupted. Inhibition was shown to occur during early infection, suggestive of involvement in virus entry or reverse transcription. In addition, we have shown that treatment with CD63 siRNA post-infection, significantly inhibited virus production in supernatant, suggesting an important role for CD63 in macrophages during HIV-1 replication events occurring after proviral integration, and possibly during egress. RESULTS: In this study we used CD63 siRNA to investigate the infectivity of pseudotyped viruses (carrying an NL4-3 Env-negative luciferase backbone) in primary human macrophages. We demonstrated that lab adapted R5- and R5X4-tropic HIV-1 strains are significantly inhibited by CD63 silencing. However, the infectivity of MLV or VSV-pseudotyped strains, which enter though receptor-mediated endocytosis, is unaffected by silencing CD63. These results indicate that CD63 may support Env-mediated entry or fusion events facilitated though CD4 and CCR5. Also, antibody and siRNA-based CD63 inhibition studies indicate a potential role for CD63 following proviral integration. Further, we show that CD63 expression is key for efficient replication in primary CD4⁺ T cells, complementing our prior studies with primary human macrophages and immortalized cell lines. CONCLUSIONS: Collectively, these findings indicate that CD63 may support Env-mediated fusion as well as a late (post-integration) step in the HIV-1 replication cycle.

In vitro model of mycobacteria and HIV-1 co-infection for drug discovery.

Tuberculosis (TB) has become a global health threat in the wake of the Human Immunodeficiency Virus (HIV) pandemic and is the leading cause of death in people with HIV/AIDS. Treatment of patients with Mycobacterium tuberculosis (Mtb)/HIV co-infection is complicated by drug interactions and toxicity that present huge challenges for clinical intervention. Discovery efforts to identify novel compounds with increased effectiveness and decreased drug-drug interactions against Mtb, HIV-1, or both, would be greatly aided by the use of a co-infection model for screening drug libraries. Currently, inhibitors of Mtb are screened independently in mycobacterial cell cultures or target based biochemical screens and less often in macrophages or peripheral blood leukocytes. Similarly, HIV-1 drugs are screened in vitro independently from anti-mycobacterial compounds. Here, we describe an in vitro model where primary human peripheral blood mononuclear cells or monocyte-derived macrophages are infected with Mycobacterium bovis BCG and HIV-1, and used to evaluate drug toxicity and activity in a co-infection setting. Our results with standard compounds (e.g. Azidothymidine, Rifampicin) demonstrate the utility of this in vitro model to evaluate drug effectiveness relevant to cellular toxicity, HIV-1 replication, and intracellular mycobacterial growth, through the use of ELISA, bacterial enumeration, and multi-variate flow cytometry. This model and associated assays have great value in accelerating the discovery of compounds for use in Mtb/HIV-1 co-infected patients.

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.

Activation of NK cell granulysin by mycobacteria and IL-15 is differentially affected by HIV.

NK cells play an important role in innate immunity to mycobacteria and are a significant source of the bactericidal effector molecule granulysin. Defects in NK cells have been described in HIV-infected patients, though mechanistic studies have focused on effector molecules relevant to anti-viral, and not anti-bacterial, function. Here we used primary NK cells from healthy human donors and an in vitro system to identify the phenotype of granulysin expressing NK cells, characterize activation stimuli that regulate granulysin, and to study the immediate effects of HIV on innate activation of NK cell granulysin expression. We observe that granulysin expression is co-associated with cytotoxicity receptors (NKp46, NKG2D) known to have important function in the cytotoxic response to M.tb-infected macrophages. Granulysin expression is significantly increased following exposure to IL-15 or Mycobacterium bovis BCG, but in contrast to our previous findings with CD8(+)T cells, expression is weakly activated by IL-21. Infection of PBMC with HIV-1 suppresses NK cell induction of granulysin by IL-15, but does not impair activation by BCG. These effects of HIV-1 are associated with reduced STAT5 phosphorylation in the IL-15 activated signaling cascade. These observations suggest that HIV may impair the anti-bacterial function of NK cells and have implications for clinical use of IL-15 to augment innate cell mediated immunity in HIV+ patients.

Host factors mediating HIV-1 replication.

Human immunodeficiency virus type 1(HIV-1) infection is the leading cause of death worldwide in adults attributable to infectious diseases. Although the majority of infections are in sub-Saharan Africa and Southeast Asia, HIV-1 is also a major health concern in most countries throughout the globe. While current antiretroviral treatments are generally effective, particularly in combination therapy, limitations exist due to drug resistance occurring among the drug classes. Traditionally, HIV-1 drugs have targeted viral proteins, which are mutable targets. As cellular genes mutate relatively infrequently, host proteins may prove to be more durable targets than viral proteins. HIV-1 replication is dependent upon cellular proteins that perform essential roles during the viral life cycle. Maraviroc is the first FDA-approved antiretroviral drug to target a cellular factor, HIV-1 coreceptor CCR5, and serves to intercept viral-host protein-protein interactions mediating entry. Recent large-scale siRNA and shRNA screens have revealed over 1000 candidate host factors that potentially support HIV-1 replication, and have implicated new pathways in the viral life cycle. These host proteins and cellular pathways may represent important targets for future therapeutic discoveries. This review discusses critical cellular factors that facilitate the successive steps in HIV-1 replication.

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.

A post-entry role for CD63 in early HIV-1 replication.

Macrophages and CD4(+) lymphocytes are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in early HIV-1 replication events in both macrophages and a CD4(+) cell line. Further analysis revealed that viral attachment and cell-cell fusion were unaffected by CD63 silencing. However, CD63-depleted macrophages showed a significant decrease in the initiation and completion of HIV-1 reverse transcription, affecting subsequent events of the HIV-1 life cycle. Integration of HIV-1 cDNA as well as the formation of 2-LTR circles was notably reduced. Reporter assays showed that CD63 down regulation reduced production of the early HIV protein Tat. In agreement, CD63 silencing also inhibited production of the late protein p24. These findings suggest that CD63 plays an early post-entry role prior to or at the reverse transcription step.

Quantitative PCR used to assess HIV-1 integration and 2-LTR circle formation in human macrophages, peripheral blood lymphocytes and a CD4+ cell line.

BACKGROUND: Integration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL). RESULTS: In this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4+ cells by infecting with HIV-1SX (R5) or dual-tropic isolate HIV-189.6 (R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA. CONCLUSION: These results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.

Non-structural and nucleocapsid proteins of Punta Toro virus induce apoptosis of hepatocytes through both intrinsic and extrinsic pathways.

Punta Toro virus (PTV; family Bunyaviridae, genus Phlebovirus) causes severe hepatic damage through brisk apoptosis of hepatocytes. In the present study, two viral proteins encoded by the S segment of the viral genome, non-structural (NSs) and nucleocapsid protein (N), were examined for their roles in apoptosis. Expression of NSs in HepG2 cells led to apoptosis in 45% of transfected cells, and with N, 28%, on average. These levels represent a four- to an eightfold increase over cells transfected with the mutated protein vectors. Caspase-3, -8 and -9 activities were increased by N protein when compared with the control NC (P < 0.05), and by NSsA and NSsB, as compared to control NSsC (P < 0.01). Treatment of the transfected cells with caspase-8 or -9 inhibitors markedly decreased apoptosis. Neutralization of TNF-alpha or Fas ligand had no effect on apoptosis. These results indicate that both NSs and N are responsible for causing hepatocyte apoptosis by triggering the extrinsic caspase-8 and intrinsic caspase-9 pathways.