Alicia Rodríguez-Huete

Molecular recognition in the human immunodeficiency virus capsid and antiviral design

Many compounds able to interfere with HIV-1 infection have been identified; some 25 of them have been approved for clinical use. Current anti-HIV-1 therapy involves the use of drug cocktails, which reduces the probability of virus escape. However, many issues remain, including drug toxicity and the emergence of drug-resistant mutant viruses, even in treated patients. Therefore, there is a constant need for the development of new anti-HIV-1 agents targeting other molecules in the viral cycle. The capsid protein CA plays a key role in many molecular recognition events during HIV-1 morphogenesis and uncoating, and is eliciting increased interest as a promising target for antiviral intervention. This article provides a structure-based, integrated review on the CA-binding small molecules and peptides identified to date, and their effects on virus capsid assembly and stability, with emphasis on recent results not previously reviewed. As a complement, we present novel experimental results on the development and proof-of-concept application of a combinatorial approach to study molecular recognition in CA and its inhibition by peptide compounds.

Rationally Designed Interfacial Peptides Are Efficient In Vitro Inhibitors of HIV-1 Capsid Assembly with Antiviral Activity

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity.

Molecular Determinants of Self-Association and Rearrangement of a Trimeric Intermediate during the Assembly of a Parvovirus Capsid

The minute virus of mice (MVM) provides a simple model for the dissection of the molecular determinants of the self-assembly, stability, and dynamics of a biological supramolecular complex. MVM assembly involves the trimerization of capsid subunits in the cytoplasm; trimers are transported to the nucleus, where they suffer a conformational change and are made competent for capsid formation. Our previous study revealed that capsid assembly from trimers is dependent on stronger intertrimer interactions that are equally spaced in an equatorial belt surrounding each trimer. We have now targeted the interfaces between monomers within each trimer to identify the molecular determinants of trimerization and the rearrangement needed for capsid assembly. Twenty-eight amino acid residues per monomer were individually mutated to alanine to remove most of the stronger intersubunit interactions. The effects on trimer and capsid assembly and virus infectivity in cells were analyzed. No side chain was individually required for trimer assembly in the cytoplasm; in contrast, half of them were required to make the trimers competent for nuclear capsid assembly, even though none was close to intertrimer interfaces. These critical side chains are conserved and participate in extensive hydrophobic contacts, buried hydrogen bonds, or salt bridges between subunits. This study on MVM capsid assembly reveals that: (i) trimerization is a robust process, insensitive to removal of individual intersubunit interactions; and (ii) the rearrangement of the trimer intermediate required for capsid assembly is a global process that depends on the establishment of many interactions along the protein-protein interfaces within each trimer.

A slender tract of glycine residues is required for translocation of the VP2 protein N-terminal domain through the parvovirus MVM capsid channel to initiate infection.

Viruses constitute paradigms to study conformational dynamics in biomacromolecular assemblies. Infection by the parvovirus MVM (minute virus of mice) requires a conformational rearrangement that involves the intracellular externalization through capsid channels of the 2Nt (N-terminal region of VP2). We have investigated the role in this process of conserved glycine residues in an extended glycine-rich tract located immediately after 2Nt. Based on the virus structure, residues with hydrophobic side chains of increasing volume were substituted for glycine residues 31 or 33. Mutations had no effect on capsid assembly or stability, but inhibited virus infectivity. All mutations, except those to alanine residues which had minor effects, impaired 2Nt externalization in nuclear maturing virions and in purified virions, to an extent that correlated with the side chain size. Different biochemical and biophysical analyses were consistent with this result. Importantly, all of the tested glycine residue replacements impaired the capacity of the virion to initiate infection, at ratios correlating with their restrictive effects on 2Nt externalization. Thus small residues within the evolutionarily conserved glycine-rich tract facilitate 2Nt externalization through the capsid channel, as required by this virus to initiate cell entry. The results demonstrate the exquisite dependence on geometric constraints of a biologically relevant translocation event in a biomolecular complex.

Effects of Macromolecular Crowding on the Inhibition of Virus Assembly and Virus-Cell Receptor Recognition

Biological fluids contain a very high total concentration of macromolecules that leads to volume exclusion by one molecule to another. Theory and experiment have shown that this condition, termed macromolecular crowding, can have significant effects on molecular recognition. However, the influence of molecular crowding on recognition events involving virus particles, and their inhibition by antiviral compounds, is virtually unexplored. Among these processes, capsid self-assembly during viral morphogenesis and capsid-cell receptor recognition during virus entry into cells are receiving increasing attention as targets for the development of new antiviral drugs. In this study, we have analyzed the effect of macromolecular crowding on the inhibition of these two processes by peptides. Macromolecular crowding led to a significant reduction in the inhibitory activity of: 1), a capsid-binding peptide and a small capsid protein domain that interfere with assembly of the human immunodeficiency virus capsid, and 2), a RGD-containing peptide able to block the interaction between foot-and-mouth disease virus and receptor molecules on the host cell membrane (in this case, the effect was dependent on the conditions used). The results, discussed in the light of macromolecular crowding theory, are relevant for a quantitative understanding of molecular recognition processes during virus infection and its inhibition.

Systematic Study of the Genetic Response of a Variable Virus to the Introduction of Deleterious Mutations in a Functional Capsid Region

ABSTRACT We have targeted the intersubunit interfaces in the capsid of foot-and-mouth disease virus to investigate the genetic response of a variable virus when individual deleterious mutations are systematically introduced along a functionally defined region of its genome. We had previously found that the individual truncation (by mutation to alanine) of 28 of the 42 amino acid side chains per protomer involved in interactions between capsid pentameric subunits severely impaired infectivity. We have now used viral RNAs individually containing each of those 28 deleterious mutations (or a few others) to carry out a total of 96 transfections of susceptible cells, generally followed by passage(s) of the viral progeny in cell culture. The results revealed a very high frequency of fixation in the capsid of second-site, stereochemically diverse substitutions that compensated for the detrimental effect of primary substitutions at many different positions. Most second-site substitutions occurred at or near the capsid interpentamer interfaces and involved residues that are spatially very close to the originally substituted residue. However, others occurred far from the primary substitution, and even from the interpentamer interfaces. Remarkably, most second-site substitutions involved only a few capsid residues, which acted as “second-site hot spots.” Substitutions at these hot spots compensated for the deleterious effects of many different replacements at diverse positions. The remarkable capacity of the virus to respond to the introduction of deleterious mutations in the capsid with the frequent fixation of diverse second-site mutations, and the existence of second-site hot spots, may have important implications for virus evolution.

Amino Acid Side Chains Buried along Intersubunit Interfaces in a Viral Capsid Preserve Low Mechanical Stiffness Associated with Virus Infectivity

Single-molecule experimental techniques and theoretical approaches reveal that important aspects of virus biology can be understood in biomechanical terms at the nanoscale. A detailed knowledge of the relationship in virus capsids between small structural changes caused by single-point mutations and changes in mechanical properties may provide further physics-based insights into virus function; it may also facilitate the engineering of viral nanoparticles with improved mechanical behavior. Here, we used the minute virus of mice to undertake a systematic experimental study on the contribution to capsid stiffness of amino acid side chains at interprotein interfaces and the specific noncovalent interactions they establish. Selected side chains were individually truncated by introducing point mutations to alanine, and the effects on local and global capsid stiffness were determined using atomic force microscopy. The results revealed that, in the natural virus capsid, multiple, mostly hydrophobic, side chains buried along the interfaces between subunits preserve a comparatively low stiffness of most (S2 and S3) regions. Virtually no point mutation tested substantially reduced stiffness, whereas most mutations increased stiffness of the S2/S3 regions. This stiffening was invariably associated with reduced virus yields during cell infection. The experimental evidence suggests that a comparatively low stiffness at S3/S2 capsid regions may have been biologically selected because it facilitates capsid assembly, increasing infectious virus yields. This study demonstrated also that knowledge of individual amino acid side chains and biological pressures that determine the physical behavior of a protein nanoparticle may be used for engineering its mechanical properties.

Different functional sensitivity to mutation at intersubunit interfaces involved in consecutive stages of foot-and-mouth disease virus assembly

Small spherical viruses are paradigms of supramolecular self-assembly. Identifying the specific structural determinants for virus assembly provides guidelines to develop new antiviral drugs or engineer modified viral particles for medical or technological applications. However, very few systematic studies have been carried out so far to identify those chemical groups at interfaces between virus capsid subunits that are important for viral assembly and function. Foot-and-mouth disease virus (FMDV) and other picornaviruses are assembled in a stepwise process in which different protein-protein interfaces are formed: 5 protomeric subunits oligomerize to form a pentameric intermediate, and 12 of these stable pentameric building blocks associate to form a labile capsid. In this study, a systematic mutational analysis revealed that very few amino acid side chains involved in substantial interactions between protomers within each pentamer are individually required for virus infectivity. This result contrasts sharply with the previous finding that most amino acid side chains involved in interactions between pentamers during the next assembly step are individually required for infectivity. The dramatic difference in sensitivity to single mutations between the two types of protein-protein interfaces in FMDV is discussed in terms of possible structural strategies for achieving self-assembly and genome uncoating in the face of diverse selective constraints.

Association equilibrium of the HIV-1 capsid protein in a crowded medium reveals that hexamerization during capsid assembly requires a functional C-domain dimerization interface.

Polymerization of the intact capsid protein (CA) of HIV-1 into mature capsidlike particles at physiological ionic strength in vitro requires macromolecularly crowded conditions that approach those inside the virion, where the mature capsid is assembled in vivo. The capsid is organized as a hexameric lattice. CA subunits in each hexamer are connected through interfaces that involve the CA N-terminal domain (NTD); pairs of CA subunits belonging to different hexamers are connected through a different interface that involves the C-terminal domain (CTD). At physiological ionic strength in noncrowded conditions, CA subunits homodimerize through this CTD-CTD interface, but do not hexamerize through the other interfaces (those involving the NTD). Here we have investigated whether macromolecular crowding conditions are able to promote hexamerization of the isolated NTD and/or full-length CA (with an inactive CTD-CTD interface to prevent polymerization). The oligomerization state of the proteins was determined using analytical ultracentrifugation in the absence or presence of high concentrations of an inert macromolecular crowding agent. Under the same conditions that promoted efficient assembly of intact CA dimers, neither NTD nor CA with an inactive CTD-CTD interface showed any tendency to form hexamers or any other oligomer. This inability to hexamerize was observed even in macromolecularly crowded conditions. The results indicate that a functional CTD-CTD interface is strictly required for hexamerization of HIV-1 CA through the other interfaces. Together with previous results, these observations suggest that establishment of NTD-CTD interactions involved in CA hexamerization during mature HIV-1 capsid assembly requires a homodimerization-dependent conformational switching of CTD.

Structural Analysis of a Temperature-Induced Transition in a Viral Capsid Probed by HDX-MS

Icosahedral viral capsids are made of a large number of symmetrically organized protein subunits whose local movements can be essential for infection. In the capsid of the minute virus of mice, events required for infection that involve translocation of peptides through capsid pores are associated with a subtle conformational change. In vitro, this change can be reversibly induced by overcoming the energy barrier through mild heating of the capsid, but little is known about the capsid regions involved in the process. Here, we use hydrogen-deuterium exchange coupled to mass spectrometry to analyze the dynamics of the minute virus of mice capsid at increasing temperatures. Our results indicate that the transition associated with peptide translocation involves the structural rearrangement of regions distant from the capsid pores. These alterations are reflected in an increased dynamics of some secondary-structure elements in the capsid shell from which spikes protrude, and a decreased dynamics in the long intertwined loops that form the large capsid spikes. Thus, the translocation events through capsid pores involve a global conformational rearrangement of the capsid and a complex alteration of its equilibrium dynamics. This study additionally demonstrates the potential of hydrogen-deuterium exchange coupled to mass spectrometry to explore in detail temperature-dependent structural dynamics in large macromolecular protein assemblies. Most importantly, it paves the way for undertaking novel studies of the relationship between structure, dynamics, and biological function in virus particles and other large protein cages.