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Determination of the relative amounts of Gag and Pol proteins in foamy virus particles
© Cartellieri et al; licensee BioMed Central Ltd. 2005
Received: 18 April 2005
Accepted: 08 July 2005
Published: 08 July 2005
We determined the relative ratios of Gag and Pol molecules in highly purified virions of spumaretroviruses or foamy viruses (FVs) using monoclonal antibodies and bacterially expressed reference proteins. We found that the cleaved p68Gag moiety dominates in infectious FVs. Furthermore, approximate mean ratios in FV are 16:1 (pr71Gag plus p68Gag:p85RT),12:1 (p68Gag:p85RT), and 10:1 (pr71Gag plus p68Gag:p40IN). Thus, the results indicate that FVs have found a way to incorporate approximately as much Pol protein into their capsids as orthoretroviruses, despite a completely different Pol expression strategy.
One of the central features of Spumaretrovirinae, which distinguishes them from Orthoretrovirinae, is the expression of a Pol precursor protein independently of the Gag protein from a spliced mRNA [1–3]. This mechanism of Pol generation raises several interesting questions: (i) How is Pol expression regulated? (ii) How is the Pol protein incorporated into the virion? (iii) And how much Pol protein is actually present in infectious viruses? While question one has, to our knowledge, not been investigated yet, answers to question two are emerging [4, 5]. Here we tried to address question three.
Theoretical lines of argument favor the view that only a few molecules of Pol may be incorporated into a FV particle. The reverse transcriptase (RT) is the main enzymatic subunit of the Pol precursor . This enzyme has been shown to be of much higher processivity than orthoretroviral RTs [7, 8]. Therefore, it was argued that FVs probably encapsidate less of their highly active Pol protein compared to orthoretroviruses [7, 8]. Following this line of argument, it is noteworthy that the FV protease (PR) is contained within the 85 kD Pol subunit, which also bears the RT/RNaseH . However, in contrast to orthoretroviruses, the FV PR cleaves the cognate Gag protein only once prior to or during budding . Therefore, FV may need less amounts of PR enzyme than orthoretroviruses.
Furthermore, experiments aimed to elucidate the mechanism of Pol protein particle incorporation (the above raised question two) indicated that Pol interacts with specific sequences on the (pre-) genomic RNA and that RNA serves as a bridging molecule between Gag (capsid) and Pol [4, 5]. Two distinct elements on the RNA have been identified, which probably facilitate this interaction . This can be regarded as another argument in support of only trace amounts of encapsidated Pol protein.
Here we wanted to investigate the approximate relative ratio of Pol to Gag molecules in infectious virions on a biochemical level to get an estimate of the FV particle composition using the prototypic FV (PFV) as a model. We did not attempt to determine absolute numbers of Gag and Pol molecules per particle.
Prokaryotic expression and purification of viral proteins
BL21(DE3)pLys (Novagen) served as a host strain for recombinant proteins. Expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside. The proteins were purified on Ni2+-chelate columns under denaturing conditions with 6 M urea. After renaturation in dialysis buffer (150 mM NaCl, 1 mM EDTA pH 5,0, 20 mM Tric-HCL pH 7,5) the amounts of purified proteins in the eluted fractions were determined by a BCA assay (Pierce). Proteins were subjected to sodium-dodecyl-sulfate-containing 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie-blue stain. The purity was analyzed by digital imaging (Phoretix 1D Advanced Version 4.01).
Pol protein is abundant in cells lytically infected with FV
Due to the presence of five Gag and Pol molecule species of different molecular weights (pr71Gag, p68Gag, pr127Po1, p85RT, and p40IN) it was not possible to calculate exactly the respective molecule numbers present in infected cells. However, the comparison of the intensity of the lanes corresponding to Gag (pr71/p68) and Pol (pr127/p85/p40) proteins, which were detected by the MABs in the lysates, indicated that high amounts of Pol are expressed upon lytic infection in BHK-21 cells. This correlates well with the published amount of pol-specific mRNA, reported to equal the full-length or gag-specific mRNA in the bovine FV system . The ease, with which Pol proteins can be detected in FV infected cells is indicative of their relatively high expression level compared to Gag. This finding questions the theoretical assumption of only trace amounts of Pol in FV particles. Obviously, FV utilizes distinct ways to avoid overloading infected cells with Pol protein. High cellular loads of retroviral Pol proteins can be associated with cell toxicity . Although not necessary to incorporate high amounts of RT in FV particles, this abundance of FV Pol proteins in infected cells may have other yet undiscovered reasons in FV biology.
Determination of the Pol protein amounts relative to Gag in FV particles
We generated highly purified virus by consecutive centrifugation through a sucrose cushion and a linear gradient made of iodixanol. BHK-21 cells were infected with the supernatant from transfected 293T cells and cell-free virus was harvested when productive infection was ongoing, usually after 3–5 days. The supernatant was clarified from cellular debris by low-speed centrifugation and filtered through a 0.45μm pore-size filter (Sartorius). Virus was concentrated by centrifugation through a 20% sucrose cushion in TNE buffer (20 mM TRIS-HC1, pH 7.5, 150 mM NaC1, 1 mM EDTA) in a SW28 rotor (Beckman) at 25,000 rpm, 4°C for 1 hr. The sediment was resolved in Dulbecco's minimal essential medium (DMEM) and placed on a 2 ml 10–40% continuous iodixanol (OptiPrep from Axis-Shield) gradient for further virus purification. The gradient was cast in a gradient mixer (SG30 from Hoefer) the day before use. Following centrifugation in a TLS-55 rotor (Beckman) at 48,000 rpm and 4°C for 4 hrs, 200 μl fractions were taken from the top. From each fraction 30 μl were used for the determination of the refraction index, 20 μ1 for infectivity assay on BHK/LTR(PFV)lacZ cells , and l00 μl for immunoblotting.
Relative amounts of Gag and Pol proteins in foamy viruses
15.8 : 1
12.3 : 1
9.8 : 1
7.8 : 1
4.2 : 1
26.3 : 1
22.7 : 1
41.3 : 1
35.8 : 1
8.0 : 1
6.8 : 1
5.2 : 1
3.0 : 1
2.3 : 1
1.3 : 1
As detailed above, the reasons to assume that only trace amounts of Pol protein are encased in spumaretrovirus virions were hitherto largely theoretical. We provide here experimental evidence that many more Pol molecules per capsid can be found in purified FVs than was previously thought, even when taking into account that we did not determine the absolute numbers of molecules per virion, but only the relative Gag to Pol ratios. How can this finding be explained in the light of recent results in which two distinct RNA structures were identified to be essential for Pol protein incorporation into FV particles ? Firstly, with respect to this study only the minimal RNA sequence requirements for Pol protein encapsidation using subgenomic constructs have been determined, and not the relative ratios between Gag and Pol using a full-length viral genome. Secondly, it may be that the presence of the RNA domains, found to be responsible for Pol packaging, leads to the encapsidation of not only two Pol molecules per viral RNA, but of a larger complex which consists of many more protein molecules. This complex may be stabilized by protein-protein interactions between Pol and Gag, the individual Pol molecules, or a combination of both.
We are indebted to Jürgen Helbig for the determination of the IgG concentration in MAB preparations.
This study was supported by grants from the DFG to A.R. (SFB479 and RE627/6-4) and to D.L. (LI621/3-1).
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