HIV-1 inactivation by 4-vinylpyridine is enhanced by dissociating Zn(2+) from nucleocapsid protein

Abstract

Selective inactivation of critical cysteine residues in human immunodeficiency virus type one (HIV-1) was observed after treatment with 4-vinylpyridine (4-VP), with and without the membrane-permeable metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Chromatographic analysis showed that cysteines contained within nucleocapsid zinc fingers, in the context of whole virus or purified protein, were essentially unreactive, but became reactive when a chelator was included. Virus treated with 4-VP showed only a modest decrease in infectivity; after TPEN addition, nearly complete inactivation of HIV-1 occurred. Similarly, quantitation of viral DNA products from 4-VP-treated virus infections showed no significant effects on reverse transcription, but did show a 14-fold reduction in proviruses; when TPEN was added, a 10(5)-fold decrease in late reverse transcription products was observed and no proviruses were detected. Since 4-VP effectiveness was greatly enhanced by TPEN, this strongly suggests that modification of nucleocapsid zinc fingers is necessary and sufficient for HIV-1 inactivation by sulfhydryl reagents.

Fig. 1

Inactivation of HIV-1. Error bars represent the standard deviation of between two and seven samples for each point. After treatment, unreacted 4-VP and TPEN were neutralized with 4 mM GSH and 100 µM Zn²⁺, respectively. (A) Virus was treated with various concentrations of 4-VP for 24 h, then the infectious titers were determined from β-galactosidase expression of TZM-bl cells, as described in Materials and methods. Titers are reported as blue cell forming units per mL (BCFU/mL). (B) Effect of 4-VP on virus infectivity, with and without TPEN; virus inactivation was monitored over 24 h. HIV-1 was incubated with PBS (○), 50 µM TPEN (▲), 1 mM 4-VP (□), and 1 mM 4-VP+50 µM TPEN (◆), and viral titers were determined as above. For times 2 h and later, the differences between VP+TPEN and the other treatments are significant based on ANOVA and post hoc Tukey tests, with p<0.001. Medium only was used to determine background levels of blue cells, which were typically <1.6 blue cell forming units per mL (BCFU/mL).

Fig. 2

HPLC analysis of HIV-1 treated with 4-VP, with and without TPEN. Absorbance at 206 nm is shown in panels on the left. Panels on the right show enlargements of 255 and 280 nm absorbance from the same sample, depicting the elution of NC and derivatives. Virus was (A) untreated, (B) treated with 1 mM 4-VP for 18 h, (C) 4-VP+50 µM TPEN for 3 h, or (D) 4-VP+TPEN for 18 h. HIV-1 treated for 18 h with TPEN alone was equivalent to untreated virus (data not shown). The data were normalized as described in Materials and methods.

Fig. 3

HPLC analysis of purified HIV-1 NC p7 protein after 4-VP treatment. Absorbance at 206 nm is shown on the left, 260 and 280 nm absorbance on the right. (A) HIV-1 NC p7 in 10 mM Tris buffer, pH 7.5, was treated with 4-VP only (~2000×[Cys]) for 195 min or (B) with 4-VP (~1000×[Cys])+EDTA for 20 min.