Bestatin-mediated inhibition of leucine aminopeptidase may hinder HIV infection

Abstract

Bestatin, an inhibitor of leucine aminopeptidase (LAPase), significantly decreased HIV infection as reflected by a reduced number of positive immunofluorescent cells, p24 levels, reverse transcriptase activity and the number of proviral copies found in Bestatin-treated cells. Cellular and extracellular LAPase activity in infected cells was higher than the LAPase activity found in uninfected cells. However, cellular and extracellular LAPase activity as well as total protein kinase C activity was lower in Bestatin-treated cells. Conversely, the incubation of human lymphocytic HUT78 cells with LAPase promotes HIV infectivity. The possible role of LAPase in the pathophysiology of HIV was assessed by determining LAPase serum levels in HIV infected patients. LAPase activity levels were three orders of magnitude greater in sera obtained from HIV patients than those detected in sera of uninfected individuals. Although Bestatin reduced HIV infection, a moderate decrease in the reverse transcriptase activity of chronically-infected H9 human T-lymphocytic cells was observed. Based on the higher levels of LAPase present in the serum of HIV patients and on the combined inhibitory effect of Bestatin on LAPase and on protein kinase C activities, we suggest that LAPase may play an important role in the early events of HIV infection such as viral entry.

Fig. 1

Dose response effect of Bestatin on reverse transcriptase activity and p24 levels after HIV-1 infection of HUT78 cells. Bestatin decreased reverse transcriptase activity and p24 levels at a concentration range of 20–200 μg/ml. Data represent the average of three different experiments with each concentration point measured in duplicate.

Fig. 2

Time response effect of Bestatin on p24 levels and reverse transcriptase activity in HIV-1 infected HUT78 cells. Twelve hours prior to infection, HUT78 cells were incubated with Bestatin at concentrations of 20, 40, and 120 μg/ml. Following infection, cells were thereafter incubated for 7 days in the presence or absence of Bestatin maintaining the same concentrations as mentioned above. Extracellular p24 antigen (a) and reverse transcriptase activity (b) was measured as a quantitative index of viral infection. Reverse transcriptase activity in chronically infected H9 CD4⁺. T-lymphocytic cells treated with Bestatin (20–200 μg/ml) showed only a moderate decrease after 7 days of treatment, compared with control and untreated cells (c).

Fig. 2

Time response effect of Bestatin on p24 levels and reverse transcriptase activity in HIV-1 infected HUT78 cells. Twelve hours prior to infection, HUT78 cells were incubated with Bestatin at concentrations of 20, 40, and 120 μg/ml. Following infection, cells were thereafter incubated for 7 days in the presence or absence of Bestatin maintaining the same concentrations as mentioned above. Extracellular p24 antigen (a) and reverse transcriptase activity (b) was measured as a quantitative index of viral infection. Reverse transcriptase activity in chronically infected H9 CD4⁺. T-lymphocytic cells treated with Bestatin (20–200 μg/ml) showed only a moderate decrease after 7 days of treatment, compared with control and untreated cells (c).

Fig. 2

Time response effect of Bestatin on p24 levels and reverse transcriptase activity in HIV-1 infected HUT78 cells. Twelve hours prior to infection, HUT78 cells were incubated with Bestatin at concentrations of 20, 40, and 120 μg/ml. Following infection, cells were thereafter incubated for 7 days in the presence or absence of Bestatin maintaining the same concentrations as mentioned above. Extracellular p24 antigen (a) and reverse transcriptase activity (b) was measured as a quantitative index of viral infection. Reverse transcriptase activity in chronically infected H9 CD4⁺. T-lymphocytic cells treated with Bestatin (20–200 μg/ml) showed only a moderate decrease after 7 days of treatment, compared with control and untreated cells (c).

Fig. 3

Bestatin-mediated inhibition of LAP and PKC activities in HIV-1 infected and non-infected HUT78 cells. Following HIV-1 infection of HUT78 cells, there was a steady increase in both cellular and extracellular LAPase activity (a). Although both cellular and extracellular LAPase activity also increased in non-infected cells (b), this increment was significantly lower. However, in both cases LAPase activity was significantly decreased in the presence of Bestatin (a,b). HUT78 cells incubated with Bestatin also showed a significant decrease in PKC activity which otherwise remained constant during 7 days of incubation (c).

Fig. 3

Bestatin-mediated inhibition of LAP and PKC activities in HIV-1 infected and non-infected HUT78 cells. Following HIV-1 infection of HUT78 cells, there was a steady increase in both cellular and extracellular LAPase activity (a). Although both cellular and extracellular LAPase activity also increased in non-infected cells (b), this increment was significantly lower. However, in both cases LAPase activity was significantly decreased in the presence of Bestatin (a,b). HUT78 cells incubated with Bestatin also showed a significant decrease in PKC activity which otherwise remained constant during 7 days of incubation (c).

Fig. 3

Bestatin-mediated inhibition of LAP and PKC activities in HIV-1 infected and non-infected HUT78 cells. Following HIV-1 infection of HUT78 cells, there was a steady increase in both cellular and extracellular LAPase activity (a). Although both cellular and extracellular LAPase activity also increased in non-infected cells (b), this increment was significantly lower. However, in both cases LAPase activity was significantly decreased in the presence of Bestatin (a,b). HUT78 cells incubated with Bestatin also showed a significant decrease in PKC activity which otherwise remained constant during 7 days of incubation (c).