1 Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107 and 2 Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethedsa, MD 20892, USA
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Abstract |
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Keywords: cleavage signal sequences/immature virus particle/mature virus particle/virion incorporation
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Introduction |
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With the goal of containing HIV-1 replication and slowing the onset of AIDS-associated diseases in infected individuals, recent efforts have focused on the development of vaccines and new therapeutic agents (Cohen, 1998; Steinman and Germain, 1998
). Specifically, there has been considerable progress in the development of anti-viral therapeutic agents targeting the reverse transcriptase and protease enzymes which are currently being used to treat HIV-1 infected individuals (Mellors, 1996
; Moyle et al., 1998
). Although the current treatment is able to prolong the latent period of infection, the inevitable emergence of drug-resistant viruses ultimately leads to the onset of AIDS in infected individuals (Wainberg and Friedland, 1998
).
As an alternative strategy to inhibit HIV-1 replication, we showed that conserved protease cleavage sites present in the Gag and Gag-pol polyproteins, can be exploited to serve as potential signals to disrupt the maturation of the virus (Serio et al., 1997). To achieve this, we generated chimeric Vpr proteins containing an additional 10 residues comprised of the protease cleavage signal sequences, found in HIV-1NL4-3. When tested for infectivity using CD4+ cells, in vitro, the viruses containing chimeric Vpr exhibited complete to moderate levels of inhibition of replication, depending on the cleavage sequence. Since Vpr is incorporated into viral particles, we hypothesized that the presence of the chimeric Vpr as a pseudosubstrate within the virus particle is likely to lead to a competition with the processing of the authentic viral polyproteins. This may ultimately result in non-infectious virus particles due to the incomplete processing of both the Gag and Gag-pol precursor proteins.
In order to understand the mechanism involved in the inhibition of viral replication by chimeric proteins, we wished to evaluate the ability of chimeric Vpr to serve as a substrate for protease, in vitro and in virus particles. The cleavage of in vitro transcribed and translated chimeric Vpr, with recombinant HIV-1 protease, was monitored by utilizing an epitope tag assay (Serio et al., 1999). This allows us to quantitate the extent of processing by densitometry. Our results indicate that chimeric Vpr can be efficiently cleaved by HIV-1 protease.
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Materials and methods |
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The plasmid DNA expressing chimeric Vpr, containing protease cleavage recognition sequences, corresponding to the cleavage sites present in Gag and Gag-pol followed by the Flag epitope (DYKDDDDK) (Figure 1A), were constructed by PCR as described (Serio et al., 1997
). The integrity of plasmid DNA was verified by sequence analysis.
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In vitro transcription/translation of proteins
The coupled T7 transcription/translation system (Promega, Madison, WI) was used for characterizing the expression of recombinant clones. The incubation conditions were followed according to the manufacturer's instructions. In vitro translated 35S-labeled proteins were used as substrates for determining the efficiency of cleavage by protease.
Protease cleavage assay
Escherichia coli cells containing the protease coding sequences in a plasmid vector were grown and induced for expression as described (Louis, et al., 1991; Wondrak and Louis, 1996
). Cells were lysed by sonication in 20 volumes of buffer [50 mM TrisHCl, pH 8.2, 1 mM dithiothreitol (DTT) and 1 mM ethylenediaminetetraacetic acid (EDTA)]. Upon centrifugation, the insoluble protein was solubilized in buffer containing 50 mM TrisHCl, pH 8.0, 7.5 M guanidine.HCl, 5 mM DTT and 5 mM EDTA, applied to a Superdex 75 column (Pharmacia, Piscataway, NJ) and equilibrated in 50 mM TrisHCl, pH 7.5, 4 M guanidine.HCl, 5 mM EDTA and 5 mM DTT, at a flow rate of 3 ml/min. The protease in the peak fractions was further purified by reversed-phase HPLC. The protease was renatured by dialysis against 50 mM sodium acetate, followed by 20 mM sodium acetate containing 2.5 mM DTT at pH 4.5 for a period of 1 h each (Wondrak et al., 1996
; Weber et al., 1997
). Protease concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL).
To perform the cleavage reaction, chimeric Vpr proteins were transcribed and translated in half the normal reaction volume (25 µl) using 0.5 µg of plasmid DNA. The entire labeled protein mixture was added to reaction buffer (50 mM formate, 2.5 mM DTT, pH 6.0) and the volume was made up to 100 µl. An aliquot was removed to correspond to the `0' h incubation time before the addition of protease. 1 µg protease was then added to each reaction mixture and samples were incubated at 30°C. Aliquots were removed at 1, 5, 10 and 30 min and reactions were terminated by the addition of an equal volume of SDS running buffer.
Quantitation by densitometry
Films representative of the cleavage reactions were used for the quanitation of cleavage efficiency of the individual chimeric Vpr. Films were scanned and densitometry was performed using the Molecular Dynamics Image QuaNT program (Serio et al., 1999). Data obtained by OD readings of each band intensity were converted to a percentage of reactivity of the substrate protein to the antibodies against the epitope with time zero representing 100%.
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Results and discussion |
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The HIV-1 genome encodes three accessory gene products designated vif, vpr and nef that are incorporated into virus particles along with the Gag and Gag-pol precursor proteins. Among these accessory proteins, Vpr has been shown to be incorporated into virus particles in higher amounts than Vif and Nef (Liu et al., 1995; Camaur and Trono, 1996
; Emerman, 1996
; Pandori et al., 1996
; Welker et al., 1996
; Bukovsky et al., 1997
). Based on this information, we generated chimeric proteins using Vpr as the carrier of choice. The chimeric Vpr proteins generated encompass Vpr coding sequences fused at the C-terminus to nine major protease cleavage site sequences (10 amino acids), found in HIV-1NL4-3 Gag and Gag-pol precursors, followed by sequences encoding the Flag epitope (FL) (Serio et al., 1997
) (Figure 1A
). Vpr-Flag (Vpr-FL) is devoid of a protease cleavage signal, with the Flag epitope located at its C-terminus. Chimeric Vpr molecules containing a protease cleavage site, in frame to the C-terminus of Vpr, followed by the Flag epitope, were given the nomenclature reflective of the cleavage site they contain as a fusion partner. To obviate any structural constraints that may restrict the accessibility of cleavage site sequences for protease, a chimeric Vpr was generated, H17/24-FL, with a flexible hinge region (GGSSG) directly upstream of the cleavage site (Kräusslich, 1991
). Expression of all the chimeric Vpr proteins was performed using an in vitro transcription/translation system, followed by immunoprecipitation with Flag antisera. The results generated are shown in Figure 2
and reveal that all proteins containing the Flag epitope are expressed, while wild-type Vpr, devoid of a Flag epitope and control plasmid pCDNA3 are not detected. In comparison with Vpr-FL, chimeric Vpr exhibited altered mobility due to the addition of amino acid residues corresponding to the cleavage site.
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Several methods have been employed to monitor cleavage of peptide and protein substrates by protease (Tözèr et al., 1991; Carter and Zybarth, 1994; Konvalinka et al., 1995
). HPLC and spectrophotometry have generally been used for the analysis of peptide substrates and antisera with specificity to the substrate and cleavage products has been used for the analysis of precursor proteins. Considering the size of chimeric Vpr (114 amino acids), we developed a new way to measure protease cleavage by using an epitope tag (Chubet and Brizzard, 1996
). A schematic diagram of the protease cleavage assay is shown in Figure 1B
. When the 35S-labeled chimeric Vpr substrates are incubated with recombinant HIV-1 protease, two cleavage products will appear. The largest product represents the entire region of the Vpr protein containing five residues upstream from the cleavage site (P5P1). The other smaller product contains five downstream residues from the cleavage site (P1'P5'), followed by the Flag epitope. After immunoprecipitating the reaction mixture, containing both the cleaved and uncleaved products, with Flag antisera, only uncleaved substrate will be recognized. By using 12% SDSPAGE, one will not be able to view the small 13 amino acid (aa) cleavage product and the larger 101 aa product, which no longer has the Flag epitope, will not be detected by Flag antisera. Since Vpr-FL lacks protease cleavage/recognition sequences, detection by Flag antisera will remain constant upon incubation with protease. Likewise, two control substrates based on the 24/2 site (KARVL-AEAMS), 24/2 D:D-FL (KARVD-DEAMS) and 24/2 G:G-FL (KARVG-GEAMS), have amino acid substitutions in the P1 and P1' positions in the original cleavage site, thereby preventing hydrolysis by protease (Dunn et al., 1994
). Reactivity of Flag antisera towards these non-hydrolyzable proteins is predicted to remain constant throughout the reaction. We are therefore able to monitor the cleavage of the chimeric Vpr substrates over time, reflected by the loss in detection, as the Flag epitope is cleaved off the substrate.
Susceptibility of chimeric Vpr to cleavage by HIV-1 protease
The cleavage of each individual chimeric Vpr was monitored over a time period of 30 min. For the sake of convenience, the results regarding the protease cleavage analysis are presented in three panels in Figure 3. Upon incubation with HIV-1 recombinant protease, chimeric Vpr proteins corresponding to the Gag cleavage sites were cleaved differentially in the 30 min time period (Figure 3A
). The chimeric Vpr protein corresponding to the 24/2 site was almost fully processed by 10 min of incubation, whereas the others still had observable substrate at times up to 30 min. The introduction of the flexible hinge into the chimeric Vpr based on 17/24, H17/24-FL, shows that cleavage can be enhanced. Additionally, chimeric Vpr containing the 7/1 and 1/6 cleavage sequences, 7/1-FL and 1/6-FL, appear to be processed less efficiently than the other Gag-derived sites in chimeric Vpr proteins.
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Quantitation of chimeric Vpr cleaved by protease
In order to characterize the extent of cleavage for each chimeric Vpr substrate, we performed densitometric analysis on the bands representing unprocessed substrate (Figure 3). Data obtained by OD readings, using the Molecular Dynamics Image QuaNT program, were converted to a percentage of reactivity of the substrate protein to the antibodies against the epitope with time zero representing 100%. The results are shown in Figure 4
. This analysis verifies that the control, chimeric Vpr proteins, are not processed by protease (Figure 4A and B
), which remain 100% reactive to Flag antisera. At 1 min incubation, chimeric Vpr representing the Gag cleavage sites remain 8633% reactive to Flag antisera. However, at 30 min they end at between 0 and 23% reactive to Flag antisera, with 24/2-FL being completely processed and H17/24-FL remaining less than 1% reactive to Flag antisera. 17/24-FL, which does not contain the flexible hinge upstream of the cleavage site (Figure 1A
), remains 13.4% reactive at 30 min, suggesting that the hinge region on H17/24-FL is allowing protease greater accessibility to the cleavage site.
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Previous work on the processing of peptide substrates and protein precursors has alluded to there being a concise order of processing of protease substrates (Erickson-Viitanen et al., 1989; Tözèr et al., 1991; Pettit et al., 1994
; Weigers et al., 1998
). Data in this area vary greatly, owing mostly to the experimental system used for analysis. Recently, work by Weigers et al. (1998) in the context of viral particles and by Pettit et al. (1994) in the context of polyproteins revealed that sequential processing is controlled by the rates of cleavage at particular sites around p2 and the initial cleavage occurs at p2/p7, followed by cleavage at 17/24, 7/1 and lastly at 24/2. Our hydrolysis results reveal that 24/2-FL and H17/24-FL are the most efficient sites for cleavage by the protease in vitro. This may be due to our chimeric Vpr containing the cleavage signal in a context outside the natural substrate, which is consistent with earlier reports showing that upstream determinants in the Gag polyproteins affect sequential cleavage (Carter and Zybarth, 1994
). Based on the extent of hydrolysis, the chimeric proteins can be grouped into three categories (Table I
). Under the conditions used, the least favorable substrate is RT/RN-FL. 24/2-FL, H17/24-FL, 2/7-FL, PR/RT-FL and 17/24-FL serve as efficient substrates. 7/1-FL, 1/6-FL, RT/IN-FL and TF/PR-FL showed moderate levels of cleavage by protease.
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Based on the data generated with chimeric Vpr synthesized in vitro, we then wanted to examine the status of chimeric Vpr, in the context of virus particles. The results from such a study will further verify the suitability of chimeric Vpr as a substrate for protease within the virion. Studies carried out by our laboratory and others have shown that the addition of residues at the C-terminus of Vpr did not alter the incorporation of Vpr into the virus particles (Wu et al., 1996, 1997
; Fletcher et al., 1997
; Serio et al., 1997
; Kobinger et al., 1998
; Okui et al., 1998
). To analyze the chimeric Vpr present within the virus particles, we have relied on a cotransfection method that has been utilized by ourselves and several other investigators (Fletcher et al., 1997
; Serio et al., 1997
; Wu et al., 1997
; Kobinger et al., 1998
). As HIV-1 proviral DNA designated NL4-3 contains Vpr coding sequences, it was reasoned that there may be competition between the Vpr expressed in the context of proviral DNA and the Vpr expressed through a heterologous promoter in a plasmid vector. To eliminate this possibility, NL4-3 was modified (NL4-3
Vpr) such that the ability to synthesize Vpr was abolished. The virus particles generated through cotransfection of NL4-3
Vpr and chimeric Vpr expression plasmids were subjected to centrifugation followed by immunoblot analysis using antiserum against the Flag epitope. The results showed that virus particles incorporated chimeric Vpr (Figure 5
). Strikingly, the reactivities of individual chimeric Vpr to Flag antiserum varied. As expected, chimeric Vpr-FL showed an intense band in comparison with other chimeric proteins. This is probably due to the fact that only Vpr-FL lacks protease cleavage signal sequences and is not hydrolyzed by the protease. Based on this, the observed signal may correspond to the overall level of incorporation of Vpr-FL into the virus particles. This is not the case, however, with other chimeric Vpr as these proteins are cleaved by protease within the virus particles and is in accord with the data noted for synthetic peptides corresponding to different cleavage sites present in the precursor proteins (Tözèr et al., 1991). These results suggest that chimeric Vpr proteins serve as substrates for protease and justify further exploratory studies on this line of investigation.
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Notes |
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Acknowledgments |
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References |
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Received October 7, 1999; revised March 1, 2000; accepted April 12, 2000.