- Short report
Mutation in the loop C-terminal to the cyclophilin A binding site of HIV-1 capsid protein disrupts proper virus assembly and infectivity
Retrovirologyvolume 4, Article number: 19 (2007)
We have studied the effects associated with two single amino acid substitution mutations in HIV-1 capsid (CA), the E98A and E187G. Both amino acids are well conserved among all major HIV-1 subtypes. HIV-1 infectivity is critically dependent on proper CA cone formation and mutations in CA are lethal when they inhibit CA assembly by destabilizing the intra and/or inter molecular CA contacts, which ultimately abrogate viral replication. Glu98, which is located on a surface of a flexible cyclophilin A binding loop is not involved in any intra-molecular contacts with other CA residues. In contrast, Glu187 has extensive intra-molecular contacts with eight other CA residues. Additionally, Glu187 has been shown to form a salt-bridge with Arg18 of another N-terminal CA monomer in a N-C dimer. However, despite proper virus release, glycoprotein incorporation and Gag processing, electron microscopy analysis revealed that, in contrast to the E187G mutant, only the E98A particles had aberrant core morphology that resulted in loss of infectivity.
The HIV-1 capsid protein (CA, p24) is the building block of the conical core structure of the virus. It is initially produced as a part of the Gag precursor (p55) and during or concomitant with the virus release, p55 is cleaved sequentially into the matrix (MA; p17), capsid, nucleocapsid (NC; p7) and p6 proteins [1, 2]. Capsid protein consists of two independently folded globular domains, the N-and C-terminal domain  connected through a short flexible hinge region.
Several studies have shown that mutations within the gag gene disrupt virus replication or infectivity [4–8] and the infectivity of HIV-1 is critically dependent on proper CA assembly and disassembly following cell entry . Although much of the assembly properties of HIV-1 CA were based on x-ray crystallographic data, NMR and in vitro assembly models, the importance of major homology region , the binding site for cyclophilin A (CypA) [11, 12], and the CA dimer interfaces [13, 14] are some of the functions in CA that have been characterised using mutational analysis.
Most of amino acid sequences in the CypA-binding loop of HIV-1 CA have been investigated using both genetic and structural studies [12, 15–17]. However, Glu98 which is well conserved  among all major HIV-1 subtypes was not previously investigated. Glu98 is located on a surface C-terminal to the CypA-binding site and has no intra-molecular contacts with other residues except for a single hydrogen bond with Arg100 . In sharp contrast, Glu187 has extensive contacts with eight other CA residues (Fig 1A and 1B).
In this study, we investigated the effects associated with two single amino acid substitution mutations, the E98A and E187G respectively, having quite opposite intra molecular CA contacts with other CA residues. The point mutations were engineered by site-directed mutagenesis and as the identity of each mutant was confirmed by sequencing, we assayed the viral protein expression using HeLa-tat and 293T cells [see Additional file 1 for details on Materials & Methods]. We found that the Western blot banding pattern of both mutants were identical to that of wild-type pNL4-3 transfected cells (Fig. 1C). Thus, the mutations did not appear to influence the intra-cellular processing of Gag precursor. We determined by ELISA that cells transfected with the E98A mutant released approximately 15% higher p24 than cells transfected with the control vector (Fig. 2A), indicating that the mutations had no substantial effect on particle release. To determine the viral protein contents of both mutant virions, viral supernatants were concentrated and separated on SDS-PAGE (Fig. 1E and 1F). The samples were then analyzed by immunoblotting with anti-glycoprotein (Fig. 1E) and a pool of HIV positive human sera (Fig. 1F). We observed that virion release was un-affected in both E98A and E187G mutants, as judged by the presence of the intermediate and fully processed Gag proteins [1, 2].
However, in contrast to the E187G and wild-type, we found that the E98A virions were non-infectious in permissive CD4 positive H9 cells (Fig. 2B), despite being competent for particle assembly, normal processing of Gag and incorporating viral envelope glycoproteins. Similar results were also seen with infected MT4 cells [see Additional file 2]. The fact that WB analysis of the E98A mutant did not show any defect in proteolytic processing of Gag indicates that the mutation may affect the later stage of virus replication, possibly post-processing. Furthermore, the level of HIV-1 glycoprotein incorporated into the budding virus particle was similar to the wild-type control suggesting that the mutation had no effect at the entry stage of the virus replication cycle. To elaborate this notion, the ability of mutant E98A virus binding and internalization was also determined on CD4+ TZM-bl cells , essentially as described elsewhere . Briefly, cells were pre-incubated at 4°C for 1 h and exposed to equal amounts of DNaseI treated E98A or wild-type virus. Following binding at 4°C or internalization at 37°C, cells were treated or not with trypsin and the amount of cell-associated p24 was measured. We observed that mutant E98A virions could bind and internalize into the target cells, indicating that there is no defect at this level of the virus replication cycle (Fig. 2C). Similar results were also seen when the intra-cellular level of viral RNAs were measured using nested RT-PCR (Fig. 2D). In this experiment, TZM-bl cells were seeded and infected as above with two-fold virus dilutions and following internalization, cells were trypsinized, washed and total RNAs were extracted. Equal amounts of RNA were then subjected to nested RT-PCR using specific primers that amplified a 593 bp fragment of the p17 viral RNA. Consequently, in order to determine the exact step at which the viral replication cycle is affected, we used a PCR based system and analyzed the early and late gene replication steps of proviral DNA synthesis in vivo in infected cells (Fig. 3A). Infection of H9 cells was performed by addition of cell-free DNaseI-treated virus produced 3 days after transfection of 293T cells. Viral DNA production by E187G mutant virion was at a level similar to that for wild-type pNL4-3. In contrast, we found that viral DNA synthesis in cells infected with E98A virus was completely absent, suggesting that the E98A mutation interferes with an early stage in the viral replication cycle.
Surprisingly, although proviral DNAs in H9 cells infected with E98A virus were not detected, a low level of Tat-induced luciferase activity was detected in a single-cell-cycle infectivity assay with TZM-bl cells (Fig. 3B). Given the fact that Tat is critical for the HIV-1 gene expression and reverse transcription [22, 23], we investigated whether a soluble Tat protein released in to the culture supernatant was involved in this assay. To address this issue, possible soluble Tat proteins in the supernatant of transfected HeLa-tat cells was immunoprecipitated using monoclonal antibody against Tat and then tested for the infectivity (Fig. 3C). However, we were unable to inhibit the subtle amount of Tat-induced luciferase activity seen in these cells and subsequently explain this activity. A possible reason may be that Tat is packaged into HIV-1 particles through binding to TAR element [24, 25], although the presence of Tat in virion has never been reported satisfactorily. Consistent with a previous report , we were also unable to detect Tat proteins in Viraffinity concentrated viral lysate using WB analysis with Tat-specific monoclonal antibody.
Since the E98A mutation is located C-terminal to the CypA-binding site and CypA has been suggested to disrupt CA-CA interactions following cell entry of the virus, we tested whether the reason for the diminished viral replication may be due to the lack of CypA incorporation in to the budding particle. However, analysis of virion-associated proteins revealed similar levels of CypA incorporation as in the control virus (Fig. 3D).
We then examined the morphological structures of these virions and correlated the results to their relative infectivity (Fig. 4). EM images of the three types of particles (NL4-3, E98A, and E187G) were categorized by the presence of three different core structures: aberrant, immature, and mature dense conical structure. Detail morphological analysis was also performed in order to depict different categories of virus morphology [see Additional file 3]. Although small percentage of virus with aberrant core was present, the majority of EM images of NL4-3 and E187G showed a mixture of both mature particles of normal morphology and immature particles (Fig. 4D). In contrast, images of E98A showed mostly aberrant and immature particles (Fig. 4B and 4D). The increased percentage (Fig. 4D) of distorted, aberrant and immature-like E98A virus particles as compared to the wild-type control may thus suggest that the E98 is important for proper protein conformation that is necessary for intermolecular CA-CA interactions. Based on the analysis of inter-atomic contacts , we found that the E98 residue is not involved in any inter-atomic contact with other CA residues. Therefore, it is possible to speculate that the E98A mutation may rather be involved in inter-molecular CA-CA interaction or with other possible cellular factors involved in this process.
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We would like to thank Ákos Végvari for critical reading and helpful discussions of the manuscript. This work was supported by grants from the Swedish Medical Research Council (grant no. K2000-06X-09501-10B), Swedish International development Cooperation Agency, SIDA (grant no. 2006-0011786) and Tripep AB
The author(s) declare that they have no competing interests.
SA performed most of the experimental work and also wrote the manuscript. SH carried out the electron microscopy analysis. AH assisted SH in electron micrograph analysis and also participated in preparing the illustrations in Figure 1. AV is the principal investigator, conceived of the study, supervised SA and wrote the manuscript together with SA. All authors read and approved the manuscript.