doi:10.1016/j.bbrc.2007.09.046 
Copyright © 2007 Elsevier Inc. All rights reserved.
Dynein light chain 1 peptide inhibits human immunodeficiency virus infection in eukaryotic cells
Danielle Renee Fontenot1, a, Petra den Hollander1, a, Eric Michael Velaa, Robert Newmana, Jagannadha K. Sastrya and Rakesh Kumar
, a, 
aUniversity of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA
Received 24 August 2007.
Available online 21 September 2007.
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Abstract
Human immunodeficiency virus (HIV) uses kinases such as Pak1 and macropinocytosis for a productive infection. Recently dynein light chain 1 (DLC1), a component of the dynein motor, was identified as a Pak1 substrate and interacted with the C-terminal region of DLC1 (aa 61–89). The dynein motor is implicated in retrograde transport, also of HIV, to the nucleus. It is known that DLC1 is important in macropinocytosis, and anti-dynein antibodies inhibit a productive HIV infection. Here, we show that in Hela-β-gal cells macropinocytosis was effectively blocked by a peptide spanning the C-terminal 19 amino acids of DLC1. We also found that the DLC1 peptide was capable of inhibiting the early entry steps of HIV, and the DLC1 peptide efficiently inhibited a productive HIV infection, and cooperated with the anti-HIV activity of CD4 antibodies. Taken together, the potentially therapeutic DLC1 peptide represents an interesting class of HIV inhibitors, targeting an essential cellular component for HIV infection. Our findings raise the possibility that the use of a DLC1 peptide in combination with currently used anti-HIV agents, might offer additional arsenal against HIV infection in human cells.
Keywords: DLC1; Human immunodeficiency virus; Macropinocytosis
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Fig. 1. DLC1 peptide inhibits macropinocytosis in HeLa-β-gal cells. (A) HeLa-β-gal cells are capable of macropinocytosis of dextran beads. Cells were serum starved for 24 h, restimulated with serum for 15 min and treated with 0.5 μg/ml FITC-labeled dextran beads for 30 min. Cells were fixed with 4% paraformaldehyde for 20 min, and counterstained with phalloidin for F-actin (red) and TOPRO3 for DNA (blue). (B) DLC peptide blocks macropinocytosis in HeLa-β-gal cells. After 24 h of serum starvation DLC peptide was applied to the cells for 45 min, cells were then stimulated with serum for 15 min, and the cells were then treated with 0.5 μg/ml of FITC-dextran for 30 min. Cells were fixed and counterstained with TOPRO3 for DNA (blue). (C) Pak1 phosphorylates DLC1, but not DLC2, and DLC1 phosphorylation can be inhibited by the DLC1 peptide. The ability of DLC1 peptide to interfere with Pak1 phosphorylation of DLC1 was assayed in the presence of 5 μg of DLC1 or control peptide. (D) Trypan blue counts show no significant difference in the cell viability when treated with increasing amounts of DLC1 peptide. Cells were plated in a 24-well plate, and after a treatment for 48 h assayed for cell viability by the standard trypan blue exclusion method.
Fig. 2. Dose-dependent inhibition of HIV productive infection by DLC1 peptide. (A) HeLa-β-gal cells were pretreated with increasing concentrations of DLC1 peptide for 1 h before live HIV virus was added. Individual infected cells were identified by staining with X-gal and counted by visual inspection. Numbers on columns represent % inhibition of HIV infection. (B) DLC1 peptide does not affect MT biogenesis. HeLa-β-gal cells were treated with 2 μg/ml of DLC1 peptide, treated with nocodazole for 30 min, washed twice with PBS and incubated with medium containing 10% FBS for 15 min. Cells were fixed in methanol and stained for tyrosinated α-tubulin. The DLC1 peptide was detected with rhodamine-labeled streptavidin.
Fig. 3. DLC1 peptide inhibits HIV RT activity. (A) MT4 assay: graph of inhibition versus protection. MT4 cells were pretreated with increasing concentrations of DLC1 peptide for 30 min before live virus was added. After 7 days the supernatant was harvested for RT analysis and cells were stained and the absorbency was measured at 590 nm on an ELISA reader to determine cell viability. DLC1 peptide treatment reduced cell death in a dose-dependent fashion. (B) RT assay: RT activity versus inhibition. Supernatant harvested from MT4 cells was assayed for reverse transcriptase activity. DLC1 peptide treatment reduced reverse transcriptase activity in a dose-dependent fashion.
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Fig. 4. DLC1 peptide inhibits HIV infection by blocking early steps of uptake. (A) DLC1 peptide was used to downregulate HIV entry via macropinocytosis. Anti-CD4-antibody treatment was used to downregulate receptor-mediated entry of HIV. The inhibitor of endosome/lysosome acidification BFLA1 was used to increase productive infection of HIV. (B) DLC1 peptide interferes with HIV-1 entry. Cells were incubated with BFLA1 prior to the addition of HIV-1. The second bar represents cells that have been treated with the DLC1 peptide prior to the addition of HIV-1 at 4 °C. The third bar represents cells that have been incubated with HIV-1 at 4 °C and then treated with DLC1 peptide. (C) PCR of HIV gene presence. PCR analysis of DLC1 peptide treated and untreated HeLa-β-gal cells for the detection of the HIV-1 gag and β-actin sequences was carried out. An HIV-1 gag band at 115 bp is observed in lanes 3 and 4. No bands are observed in lanes 1 and 2, cells pretreated with 2 and 10 μg/ml concentration of the DLC1 peptide.
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