Jason T. Blackard

Extrahepatic replication of HCV: Insights into clinical manifestations and biological consequences†

An estimated 170 million persons are infected with the hepatitis C virus (HCV) worldwide. While hepatocytes are the major site of infection, a broad clinical spectrum of extrahepatic complications and diseases are associated with chronic HCV infection, highlighting the involvement of HCV in a variety of non‐hepatic pathogenic processes. There is a growing body of evidence to suggest that HCV can replicate efficiently in extrahepatic tissues and cell types, including peripheral blood mononuclear cells. Nonetheless, laboratory confirmation of HCV replication in extrahepatic sites is fraught with technical challenges, and in vitro systems to investigate extrahepatic replication of HCV are severely limited. Thus, future studies of extrahepatic replication should combine innovative in vitro assays with a prospective cohort design to maximize our understanding of this important phenomenon to the pathogenesis and treatment response rates of HCV. (HEPATOLOGY 2006;44:15–22.)

Hepatitis C Virus Core Protein Blocks Interferon Signaling by Interaction with the STAT1 SH2 Domain

ABSTRACT Emerging data have indicated that hepatitis C virus (HCV) subverts the host antiviral response to ensure its persistence. We previously demonstrated that HCV protein expression suppresses type I interferon (IFN) signaling by leading to the reduction of phosphorylated STAT1 (P-STAT1). We also demonstrated that HCV core protein directly bound to STAT1. However, the detailed mechanisms by which HCV core protein impacts IFN signaling components have not been fully clarified. In this report, we show that the STAT1 interaction domain resides in the N-terminal portion of HCV core (amino acids [aa] 1 to 23). This domain is also required to produce P-STAT1 reduction and inhibit IFN signaling transduction. Conversely, the C-terminal region of STAT1, specifically the SH2 domain (aa 577 to 684), is required for the interaction of HCV core with STAT1. The STAT1 SH2 domain is critical for STAT1 hetero- or homodimerization. We propose a model by which the binding of HCV core to STAT1 results in decreased P-STAT, blocked STAT1 heterodimerization to STAT2, and, therefore, reduced IFN-stimulated gene factor-3 binding to DNA and disrupted IFN-stimulated gene transcription.

Human Immunodeficiency Virus Superinfection and Recombination: Current State of Knowledge and Potential Clinical Consequences

Superinfection with multiple strains or subtypes of the human and simian immunodeficiency viruses has been documented. Recent increases in the prevalences of both unprotected anal intercourse and sexually transmitted diseases among men who have sex with men indicate that these men continue to practice unsafe sex and, therefore, are at risk for superinfection with the human immunodeficiency virus (HIV). Recurrent exposure to HIV among seropositive individuals who engage in high-risk behaviors can have serious consequences, because superinfection is a necessary first step for viral recombination to occur. Recombination may produce more virulent viruses, drug-resistant viruses, or viruses with altered cell tropism. Additionally, recombinant viruses and superinfection can accelerate disease progression and increase the likelihood of sexual transmission by increasing virus load in the blood and genital tract. The extent of superinfection and recombination in persons living with HIV is unknown. The implications of HIV superinfection and the generation of recombinant viruses are discussed.

Acute Hepatitis C Virus Infection: A Chronic Problem

Since the discovery of the hepatitis C virus (HCV) in the late 1980s, there has been an explosion of information regarding its natural history, treatment, and replication cycle. Nonetheless, there are still relatively few data regarding acute HCV infection. By convention, the term acute hepatitis refers to the presence of clinical signs or symptoms of hepatitis for a period of 6 months or fewer after the presumed time of HCV exposure. Early studies of posttransfusion patients who developed non-A, non-B hepatitis provide a clinical picture of early infection.1 Following the availability of specific serologic and virologic tests, most such patients were shown to have acute HCV infection. After acute infection, HCV RNA may become detectable in the serum/plasma in as little as 2 weeks (Fig. 1). Several weeks later, a high percentage of patients experience an increase in serum aminotransferase levels consistent with the development of acute hepatocellular injury. In the majority of cases, patients develop mild constitutional symptoms, including abdominal pain, nausea, vomiting, anorexia, and fatigue. During this acute infection, serum aminotransferases often peak below 1000 IU/mL and may return to normal levels. A minority develops sufficient elevations in bilirubin to lead to overt jaundice or the development of dark urine. Unless the clinical suspicion is high, few patients will be tested for HCV RNA or HCV antibody seroconversion. However, in the majority— but not all— of infected patients, HCV RNA persists, and a chronic disease state develops. Fig. 1 Natural history of HCV infection (upper panel) and immunologic responses to HCV infection (lower panel): (−) HCV RNA, (- - -) cellular response, and (· · ·) ALT. ALT indicates alanine aminotransferase; HCV, hepatitis C ... The reasons for the general lack of data regarding acute HCV infection are multifactorial and include (1) the relatively high percentage of asymptomatic or unrecognized early infections, (2) the lack of large-scale identification of chronic carriers in the general population who serve as a reservoir for infection, and (3) the decreased number of acute infections that occur in controlled clinical settings such as that of blood transfusions. These factors and the lack of nonprimate animal models necessitate reliance on retrospective studies in chronic carriers, the use of limited historical collections of banked sera, and the extrapolation of outcomes based on small disease outbreaks in unique settings (for example, transmission from a physician to a patient in the operating room setting or following parenteral exposure in healthcare workers). Moreover, there exist only a limited number of population cohorts that continue to experience high rates of HCV transmission (for example, Egypt); nonetheless, there is a growing body of information regarding the clinical presentation, natural history, and treatment outcomes of acute HCV infection. In this article, we review the current information regarding our understanding of the epidemiology, virology, and immunology of HCV with a particular emphasis on acute HCV infection. In addition, we review recent data related to interferon-based treatment intervention and propose an algorithm for the diagnosis and management of acute HCV infection.

Human Regulatory T Cells Are Targets for Human Immunodeficiency Virus (HIV) Infection, and Their Susceptibility Differs Depending on the HIV Type 1 Strain

ABSTRACT Regulatory T cells (Treg) are a subpopulation of CD4+ T cells characterized by the suppressive activity they exert on effector immune responses, including human immunodeficiency virus (HIV)-specific immune responses. Because Treg express CXCR4 and CCR5, they represent potential targets for HIV; however, Treg susceptibility to HIV infection is still unclear. We therefore performed an extensive study of Treg susceptibility to HIV, using lab strains and primary isolates with either CCR5 or CXCR4 tropism. Furthermore, we quantified HIV infection at early and late time points of the virus life cycle. We found that Treg were clearly susceptible to HIV infection. Circulating Treg were not preferentially infected with HIV compared to effector T cells (Teff) in vivo. Conversely, in vitro infection with either CCR5-using (R5) or CXCR4-using (X4) viruses occurred with different dynamics. For instance, HIV infection by R5 viruses (lab strains and primary isolates) resulted in lower levels of infection in Treg compared with Teff at both early and late time points. In contrast, X4 viruses induced higher levels of infection in Treg compared to Teff at early time points, but this difference disappeared at the late time points of the virus life cycle. Our results suggest that the relative susceptibility of Treg to HIV infection compared to Teff varies, depending on both viral and host factors. These variations may play an important role in HIV pathogenesis.

Detection of Hepatitis C Virus (HCV) in Serum and Peripheral-Blood Mononuclear Cells from HCV-Monoinfected and HIV/HCV–Coinfected Persons

It has been speculated that hepatitis C virus (HCV) replicates in peripheral-blood mononuclear cells (PBMCs), which, therefore, may be a site for interaction with human immunodeficiency virus (HIV). We used strand-specific real-time polymerase chain reaction to detect HCV RNA in 28 HCV-monoinfected and 20 HIV/HCV-coinfected women. At the first visit, positive-strand HCV RNA was detected in serum samples from 89% of the women, whereas positive-strand HCV RNA was detected in PBMC samples from 32% and 55% of the HCV-monoinfected and HIV/HCV-coinfected women, respectively. After initiation of antiretroviral therapy, the HIV/HCV-coinfected women were significantly more likely to have detectable positive- and negative-strand HCV RNA in the PBMC compartment than were the HCV-monoinfected women. HIV and HCV RNA levels were not correlated. Serum HCV RNA levels were correlated over time; HCV RNA levels in the serum and PBMC compartments were not. These data suggest differential regulation of HCV RNA in the serum and PBMC compartments and may partially explain the limited HCV antiviral response rates observed in coinfected persons.

HCV/ HIV co-infection: time to re-evaluate the role of HIV in the liver?

Summary.  Because of major advances in the treatment of HIV/AIDS, HIV‐positive persons now live longer, healthier lives; however, hepatitis C virus (HCV) is increasingly recognized as a major cause of morbidity and mortality in this population. Among HCV‐infected persons, HIV co‐infection is associated with increased HCV RNA levels, increased hepatic inflammation and fibrosis, and more rapid progression to cirrhosis and end‐stage liver disease. Compounding this problem are reduced HCV treatment response rates among HCV/HIV co‐infected persons. Moreover, antiretroviral therapy used to suppress HIV replication is often associated with a paradoxical increase in HCV RNA levels, as well as hepatotoxicity. Despite the adverse clinical consequences of HCV/HIV co‐infection, the mechanisms by which these two viruses interact at the cellular level remain largely unexplored. This review focuses on the evidence demonstrating direct infection of hepatocytes by HIV, as well as the indirect mechanisms by which HIV may regulate HCV replication at the cellular level. A comprehensive understanding of virus–virus and virus–cell interactions is critical to the development of novel treatment strategies to combat HCV/HIV co‐infection.

Hepatitis C virus acts as a tumor accelerator by blocking apoptosis in a mouse model of hepatocarcinogenesis

We developed hepatitis C virus (HCV) core‐E1‐E2 and HCV core transgenic mice on a common genetic background to assess the contribution of HCV structural proteins to hepatocarcinogenesis. Eight‐week‐old core‐E1‐E2, core, and nontransgenic mice inbred on the FVB×C57Bl/6 background were treated with diethylnitrosamine (DEN) and sacrificed at 32 weeks old. Proliferation and apoptosis were assessed by immunohistochemistry. The effect of viral proteins on apoptosis was evaluated in HepG2 cells in which apoptosis was induced by anti‐Fas antibody. HCCs were identified at 32 weeks in the majority of DEN‐treated mice from all three groups. The mean size of HCCs was significantly larger in core‐E1‐E2 transgenic (4.63 ± 1.48 mm), compared with core transgenic (0.78 ± 0.26 mm, P = .01), and nontransgenic (1.0 ± 0.19 mm, P = .002) mice. While there were no differences in proliferation, the apoptotic index in core‐E1‐E2 transgenic HCCs was significantly lower than those found in core and non‐transgenic HCCs. Core‐E1‐E2 transfected HepG2 cells demonstrated a significantly lower apoptotic index (0.35 ± 0.11) compared with that of core transfected cells (0.74 ± 0.07, P = .0103). Analysis of a Fas‐induced apoptosis model in HCV transgenic mice confirmed that core‐E1‐E2 transgenic liver underwent significantly less apoptosis than transgenic tissue expressing core only. In conclusion, HCV core‐E1‐E2 transgenic mice develop significantly larger tumors than transgenic mice expressing core alone or nontransgenic mice. The accelerated tumor phenotype is attributable to suppression of apoptosis rather than enhanced proliferation. These data implicate HCV E1 and/or E2 in conjunction with core as antiapoptotic, tumor accelerator proteins. (HEPATOLOGY 2005; 41:660–667.)

HCV E2 protein binds directly to thyroid cells and induces IL-8 production : A new mechanism for HCV induced thyroid autoimmunity

HCV infection is well-known to be associated with autoimmune thyroiditis. However, the mechanisms by which HCV triggers thyroiditis are unknown. We hypothesized that HCV envelope proteins could induce thyroidal inflammation directly, thereby triggering thyroiditis by a bystander activation mechanism. To test this hypothesis we examined whether the HCV receptor CD81 was expressed and functional on thyroid cells. We found significant levels of CD81 mRNA by QPCR analysis, as well as CD81 protein by flow cytometric (FACS) analysis. Incubation of thyroid cells with HCV envelope glycoprotein E2 resulted in E2 binding to thyroid cells and activation of IL-8, an important pro-inflammatory cytokine. Intriguingly, thyroid cells incubated with E2 continued to proliferate normally and did not undergo apoptosis, as was reported in hepatocytes. We conclude that: (1) HCV envelope glycoprotein E2 can bind to CD81 receptors which are expressed on thyroid cells and induce a cascade of signaling pathway leading to IL-8 release; and (2) HCV may trigger thyroiditis in genetically susceptible individuals by bystander activation mechanisms.

The prevalence and significance of occult hepatitis B virus in a prospective cohort of HIV-infected patients.

Background:Occult hepatitis B virus (HBV) is defined as low-level HBV DNA without hepatitis B surface antigen (HBsAg). Prevalence estimates vary widely. We determined the prevalence of occult HBV at the University of Cincinnati Infectious Diseases Center (IDC). Methods:Patients in the IDC HIV database (n = 3867) were randomly selected using a 25% sampling fraction. Samples were pooled for HBV nucleic acid extraction. Pools were tested for HBV DNA by a real-time polymerase chain reaction (PCR) assay to coamplify core/surface protein regions. The PCR assay was run on all individual samples from each DNA+ pool. DNA+ samples were tested for HBV serologic markers. Results:A total of 909 patients without known HBV were selected. The mean CD4 count was 384 cells/mm3. Forty-three patients were HBV DNA+. Twelve of 43 were DNA+/HBsAg− (95% confidence interval for database: 0.58% to 1.90%). Five of 12 were negative for all serologic markers. Alanine aminotransferase, aspartate aminotransferase, and HBV DNA titers were elevated in HBsAg+ patients versus occult patients and versus HIV-monoinfected patients. No other significant differences were detected. No occult HBV patient was on treatment with anti-HBV activity. Conclusions:Forty-three percent of those with HBV were not previously identified as HBV+, indicating the need for ongoing screening in high-risk populations. Occult HBV may occur in persons with all negative serologic markers, representing a challenge for identification.