To date, only one published study has examined HIV variability in the liver. Van't Wout et al
. assessed HIV proviral DNA variability from a single individual who died of AIDS-related complications.46
They concluded that the presence of HIV in nonlymphoid tissues was likely the result of the late disease stage of the samples examined, although samples collected from earlier disease stages were not included. Similarly, Donaldson et al
. reported that infection of nonlymphoid organs, such as the liver, occurred only in individuals with AIDS-defining illnesses and not among asymptomatic individuals11
; however, HIV variability was not explored. In contrast, in the current study, HIV RNA was detected in nine individuals with a wide range of CD4 cell counts (0 to 601 cells/mm3
) suggesting that HIV infection of the liver may occur at varying levels of immunosuppression.
Importantly, several lines of evidence suggest that the liver may represent a potential site of HIV compartmentalization. First, the presence of distinct consensus sequences in the liver compared to the corresponding plasma/PBMCs argues against simple contamination of biopsy tissues with peripheral lymphocytes and/or cell-free virions from the peripheral blood supply. Second, patient-specific clonal analysis frequently demonstrated distinct HIV variants in the liver compared to the corresponding plasma. These findings were further supported statistically by the results of both Mantel's and Slatkin–Maddison tests. Third, signature sequence analysis identified 49 amino acid residues in the region of env sequenced that were associated with HIV detection in the liver. These amino acids imply adaptation of HIV for infection of the liver and may impact HIV replication and/or cell tropism.
Despite frequent observation of liver enzyme abnormalities in those with HIV infection, HIV treatment providers rarely include liver biopsy in the evaluation process of these patients. Liver biopsy is more commonly employed in patients with HBV and/or HCV infection; therefore, few investigators have had access to samples that would permit assessment of the direct effects of HIV on the liver in vivo
. While our methodology does not provide any information regarding the specific cell type(s) that may be infected by HIV or the overall level of HIV replication in the liver, there is considerable evidence suggesting that several liver cell types can support HIV replication as reviewed elsewhere.1
Thus, it is reasonable to assume that there is at least one cell type in the liver that is capable of supporting HIV replication. Although we cannot definitively rule out contamination by PBMC-derived HIV, this is unlikely for two reasons. First, several studies have shown little or no compartmentalization, or similar mutational patterns, in PBMCs compared to plasma/serum.47–49
Second, when PBMCs were included from subject 1756, PBMC- and plasma-derived viruses grouped together but were separate from liver-derived sequences.
Several limitations of the current study warrant further discussion. While the population size is modest, these data represent the largest study of HIV variability in the liver ever performed. Additionally, while several distinct methods to detect viral compartmentalization are available, there is no one gold standard or preferred approach. For example, Zarate et al
. compared multiple methods for detecting HIV compartmentalization and found that discordant predictions by distinct methods may occur; therefore, utilizing several complementary methods, as performed here, provides the most reliable assessment of viral compartmentalization.50
Furthermore, the cross-sectional nature of this analysis does not permit a detailed examination of liver-specific HIV variants over time or provide important data on the possible trafficking of liver-specific HIVs into the peripheral circulation. Similarly, it is possible that our cloning strategy may not have amplified all minor variants present in a given tissue/cell type.
Our study is the first to explore HIV RNA variability in the liver and to demonstrate distinct HIV variants in liver biopsy tissues. However, it is important to note that HIV was not amplifiable in all samples examined, although we did not directly quantify intrahepatic levels of HIV in the current study. Thus, identification of the virologic, immunologic, and genetic factors that impact HIV detection in the liver will require additional study. Moreover, exploring the variability of additional HIV genomic regions amplified from the liver, as well as detecting low-frequency viral variants by single-genome amplification, is warranted and may enhance our understanding of HIV pathogenesis. Finally, additional studies are currently underway to determine the relative contributions of distinct liver cell types to HIV pathogenesis and viral diversity, as well as the impact of liver-derived HIV on liver damage and fibrosis progression.