Characterization of a discontinuous neutralizing epitope on glycoprotein B of human cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.

pH-dependent molecular dynamics of vesicular stomatitis virus glycoprotein G.

Vesicular stomatitis virus glycoprotein G (VSV-G) belongs to a new class of viral fusion proteins (Class III). The structure of VSV-G has been solved in two different conformations and fusion is known to be triggered by low pH. To investigate Class III fusion mechanisms, molecular dynamics simulations were performed on the VSV-G prefusion structure in two different protonation states: at physiological pH (pH 7) and low pH present in the endosome (pH 5). Domain IV containing the fusion loops, which need to interact with the target membrane, exhibits the highest mobility. Energetic analyses revealed weakened interaction between Domain IV and the protein core at pH 5, which can be attributed to two pairs of structurally neighboring conserved and differentially protonated residues in the Domain IV-core interface. Energetic calculations also demonstrated that the interaction between the subunits in the core of the trimeric VSV-G is strengthened at pH 5, mainly due to newly formed interactions between the C-terminal loop of Domain II and the N-terminus of the adjacent subunit. A pair of interacting residues in this interface that is affected by differential protonation was shown to be the main effectors of this phenomenon. The results of this study thus enhance the mechanistic understanding of the effects of protonation changes in VSV-G.

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies.

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.

A comparative study of HIV-1 and HTLV-I protease structure and dynamics reveals a conserved residue interaction network.

The two retroviruses human T-lymphotropic virus type I (HTLV-I) and human immunodeficiency virus type 1 (HIV-1) are the causative agents of severe and fatal diseases including adult T-cell leukemia and the acquired immune deficiency syndrome (AIDS). Both viruses code for a protease that is essential for replication and therefore represents a key target for drugs interfering with viral infection. The retroviral proteases from HIV-1 and HTLV-I share 31% sequence identity and high structural similarities. Yet, their substrate specificities and inhibition profiles differ substantially. In this study, we performed all-atom molecular dynamics (MD) simulations for both enzymes in their ligand-free states and in complex with model substrates in order to compare their dynamic behaviors and enhance our understanding of the correlation between sequence, structure, and dynamics in this protein family. We found extensive similarities in both local and overall protein dynamics, as well as in the energetics of their interactions with model substrates. Interestingly, those residues that are important for strong ligand binding are frequently not conserved in sequence, thereby offering an explanation for the differences in binding specificity. Moreover, we identified an interaction network of contacts between conserved residues that interconnects secondary structure elements and serves as a scaffold for the protein fold. This interaction network is conformationally stable over time and may provide an explanation for the highly similar dynamic behavior of the two retroviral proteases, even in the light of their rather low overall sequence identity.