Several lines of evidence suggest the potential for HIV to interact directly with multiple liver cell populations in vivo (reviewed in
). For instance, HIV RNA and proviral DNA have been detected in liver biopsies from persons with HIV infection
[28–30]. Subsequent immunohistochemistry and in situ hybridization studies using liver specimens from HIV-infected patients have demonstrated HIV p24 protein and HIV RNA in Kupffer cells, inflammatory mononuclear cells, sinusoidal cells, intrahepatic lymphocytes, and/or hepatocytes
[28, 31–35]. Efficient activation of the HIV long terminal repeat also occurs in hepatocytes
[36–38]. HIV compartmentalization in the liver has also been reported recently, suggesting that viral adaptation for replication within the liver may occur in vivo . Soluble HIV proteins, such as gp120, also induce caspase expression and hepatic apoptosis
[39–41], as well as interleukin 8 (IL-8), a pro-inflammatory chemokine that is an important mediator of hepatic inflammation and is known to antagonize the antiviral effects of IFN
Despite multiple studies assessing the effects of HIV proteins on hepatocytes in vitro, contradictory reports of HIV entry receptor expression
[39, 40, 46, 47] may have lessened enthusiasm for investigating the ability of HIV to infect and replicate within hepatocytes. Nonetheless, several recent reports provide evidence that HIV can productively infect hepatocytes at low levels. For example, Xiao et al. isolated a CD4-independent strain of HIV from a patient with advanced HIV disease that was capable of infecting hepatocyte cell lines, as well as primary hepatocytes
. Fromentin et al. demonstrated that Huh7.5 cells bind to and internalize HIV particles and that HIV infection of CD4+ T cells was enhanced after interacting with virus-loaded hepatocytes compared to cell-free virus
. Finally, Iser et al. observed increased HIV reverse transcriptase activity following HIV infection of hepatocyte cell lines
. While the precise receptors used by HIV to enter hepatocytes remain elusive, Iser et al. reported that, despite the inability to detect surface expression of CD4, CCR5, or CXCR4 by flow cytometry, infection with R5 or X4 HIV was inhibited by maraviroc or AMD3100, respectively, suggesting CCR5- or CXCR4-dependent entry
. We have also detected CCR1, CCR2, and CCR3 co-receptors on Huh7.5 cells (Cardona-Maya and Blackard, unpublished data). Therefore, these receptors may also contribute to HIV infection of hepatocytes, although this requires additional investigation.
The current investigation was considered a proof-of-principle study designed to assess whether HIV was capable of infecting hepatocytes or not. Thus, several cell lines (HCV infected and HCV uninfected), as well as primary hepatocytes, were utilized. This study complements previous investigations and provides several novel findings. First, the Huh7.5 and Huh7.5JFH1 cell lines, as well as primary hepatocytes, supported early steps in the HIV life cycle as demonstrated by detection of integrated HIV DNA. Second, hepatocyte cell lines and primary hepatocytes also support late steps in the HIV life cycle as demonstrated by p24 detection in the supernatants and/or lysates of infected cells and infection of an HIV indicator cell line with hepatocyte-derived HIV. This finding is particularly interesting in light of the recent study by Fromentin et al. demonstrating that circulating CD4+ T cells can be potentially infected with HIV through contact with hepatocytes
. Our data further suggest that replication of HIV in hepatocytes is occurring at relatively low levels as integrated DNA levels in hepatocytes were lower than levels in Jurkats. Similarly, supernatants from HIV-infected hepatocytes contained fewer HIV particles that were able to infect an indicator cell line than HIV-infected Jurkats. These findings are in agreement with other recent reports that hepatocytes permit low-level replication of HIV
[49, 50].The levels of HIV infection achieved in primary hepatocytes versus hepatocyte cell lines were not equivalent. This may reflect differing levels of permissiveness to HIV in primary hepatocytes versus hepatocyte cell lines, possibly due to different levels of entry receptors or innate antiviral defense molecules. Interestingly, single-cell cloning has been utilized to examine permissiveness of Huh7 cells to HCV infection
. The finding of sub-populations of the parental cell line with distinct phenotypic characteristics implies that HIV infectivity could also be impacted, although a comprehensive analysis of HIV infection of primary hepatocytes derived from distinct patients has not been performed to date. Thus, future studies that address the expression of HIV entry receptors, as well as cellular factors that restrict HIV infection should be examined in primary hepatocytes from multiple donors and correlated with overall levels of HIV replication.
We were quite interested to find that HIV could indeed infect an HCV-infected hepatocyte cell line. In a preliminary investigation, we found that HIV infection of Huh7.5JFH1 cells resulted in increased positive- and negative-sense HCV RNA levels, as well as increased HCV protein expression, compared to HIV uninfected cells
. However, HIV-induced HCV replication was abolished in the presence of antiretroviral agents. These data complement in vivo reports that HCV RNA levels are elevated in individuals with HIV/HCV co-infection compared to those with HCV mono-infection
[11, 53, 54] and suggest that the HCV-infected Huh7.5JFH1 cell line could be developed as an in vitro model to characterize mechanisms by which HIV and HCV interact at the cellular level and contribute to accelerated liver disease.
In conclusion, the present results demonstrate that the Huh7.5 and Huh7.5JFH1 cell lines, as well as primary hepatocytes, can be infected with HIV. These studies provide the necessary systems to further expand our understanding of virus-virus and virus-host interactions that are relevant for enhancing our understanding of how HIV impacts liver disease, as well as increased replication of hepatitis viruses. Moreover, these investigations could ultimately lead to optimization of current therapies for HIV and ameliorate the deleterious effects of HIV on liver disease.