Three-dimensional molecular structures can provide detailed information on biological mechanisms and, for cases in which the molecular function affects human health, can significantly aid in the ...development of therapeutic interventions. For almost 25 years, key components of the lentivirus HIV-1, including the envelope glycoproteins, the capsid and the replication enzymes reverse transcriptase, integrase and protease, have been scrutinized to near atomic-scale resolution. Moreover, structural analyses of the interactions between viral and host cell components have yielded key insights into the mechanisms of viral entry, chromosomal integration, transcription and egress from cells. Here, we review recent advances in HIV-1 structural biology, focusing on the molecular mechanisms of viral replication and on the development of new therapeutics.
Retroviral replication proceeds through a stable proviral DNA intermediate, and numerous host cell factors have been implicated in its formation. In particular, recent results have highlighted an ...important role for the integrase-interactor lens epithelium-derived growth factor (LEDGF)/p75 in lentiviral integration. Cells engineered to over-express fragments of LEDGF/p75 containing its integrase-binding domain but lacking determinants essential for chromatin association are refractory to HIV-1 infection. Furthermore, both the levels of HIV-1 integration and the genomic distribution of the resultant proviruses are significantly perturbed in cells devoid of endogenous LEDGF/p75 protein. A strong bias towards integration along transcription units is a characteristic feature of lentiviruses. In the absence of LEDGF/p75, HIV-1 in large part loses that preference, displaying concomitant integration surges in the vicinities of CpG islands and gene promoter regions, elements naturally targeted by other types of retroviruses. Together, these findings highlight that LEDGF/p75 is an important albeit not strictly essential cofactor of lentiviral DNA integration, and solidify a role for chromatin-associated LEDGF/p75 as a receptor for lentiviral preintegration complexes. By now one of the best characterized virus-host interactions, the integrase-LEDGF/p75 interface opens a range of opportunities for lentiviral vector targeting for gene therapy applications as well as for the development of novel classes of antiretroviral drugs.
To establish productive infection, a retrovirus must insert a DNA replica of its genome into host cell chromosomal DNA. This process is operated by the intasome, a nucleoprotein complex composed of ...an integrase tetramer (IN) assembled on the viral DNA ends. The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the viral and cellular DNA molecules. Although several intasome/transpososome structures from the DDE(D) recombinase superfamily have been reported, the mechanics of target DNA capture and strand transfer by these enzymes remained unclear. Here we report crystal structures of the intasome from prototype foamy virus in complex with target DNA, elucidating the pre-integration target DNA capture and post-catalytic strand transfer intermediates of the retroviral integration process. The cleft between IN dimers within the intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced IN active sites to access the scissile phosphodiester bonds. Our results resolve the structural basis for retroviral DNA integration and provide a framework for the design of INs with altered target sequences.
Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral ...immunity. SARS-CoV-2 spike glycoprotein (S)-reactive antibodies were detectable using a flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S-reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2-uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.
Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines show protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, ...spike. Because new SARS-CoV-2 variants are emerging rapidly, as exemplified by the B.1.1.7, B.1.351, and P.1 lineages, it is critical to understand whether antibody responses induced by infection with the original SARS-CoV-2 virus or current vaccines remain effective. In this study, we evaluate neutralization of a series of mutated spike pseudotypes based on divergence from SARS-CoV and then compare neutralization of the B.1.1.7 spike pseudotype and individual mutations. Spike-specific monoclonal antibody neutralization is reduced dramatically; in contrast, polyclonal antibodies from individuals infected in early 2020 remain active against most mutated spike pseudotypes, but potency is reduced in a minority of samples. This work highlights that changes in SARS-CoV-2 spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their effect on vaccine efficacy.
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•SARS-CoV-2 pseudotypes produced by amino acids substitutions in SARS-CoV•SARS-CoV-2 pseudotyped virus that encodes the B.1.1.7 variant spike•Amino acid changes and B.1.1.7 can decrease monoclonal antibody neutralization•Minimal effect on sera with only 10% losing potency against the B.1.1.7 pseudotype
This study describes neutralization by antibodies and convalescent sera of SARS-CoV-2 spike mutants. Rees-Spear et al. show that SARS-CoV amino acid substitutions and the B.1.1.7 variant can block monoclonal antibody neutralization and that serum samples collected following mild illness are less resilient to spike variation than those following severe illness.
Abstract Three-dimensional macromolecular structures shed critical light on biological mechanism and facilitate development of small molecule inhibitors. Clinical success of raltegravir, a potent ...inhibitor of HIV-1 integrase, demonstrated the utility of this viral DNA recombinase as an antiviral target. A variety of partial integrase structures reported in the past 16 years have been instrumental and very informative to the field. Nonetheless, because integrase protein fragments are unable to functionally engage the viral DNA substrate critical for strand transfer inhibitor binding, the early structures did little to materially impact drug development efforts. However, recent results based on prototype foamy virus integrase have fully reversed this trend, as a number of X-ray crystal structures of active integrase-DNA complexes revealed key mechanistic details and moreover established the foundation of HIV-1 integrase strand transfer inhibitor action. In this review we discuss the landmarks in the progress of integrase structural biology during the past 17 years.
Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and ...several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants.
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•Potent nAbs were isolated from an asymptomatic donor with low plasma neutralization•RBD-specific nAbs target epitopes overlapping with known RBD antibody classes•NTD mutations in B.1.1.7 Spike confer neutralization resistance to NTD-specific nAbs•Most RBD-specific nAbs retain potent neutralization of the B.1.1.7 variant
The impact of mutations arising in SARS-CoV-2 Spike on antigenicity is still not known. Graham et al. isolate potent neutralizing monoclonal antibodies from individuals experiencing a range of COVID-19 disease severity that target RBD, NTD, and non-S1 epitopes. The B.1.1.7 variant of concern was most resistant to NTD-specific nAbs whereas RBD-specific nAbs retained potent neutralization.
Several variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged during the current coronavirus disease 2019 (COVID-19) pandemic. Although antibody cross-reactivity with ...the spike glycoproteins (S) of diverse coronaviruses, including endemic common cold coronaviruses (HCoVs), has been documented, it remains unclear whether such antibody responses, typically targeting the conserved S2 subunit, contribute to protection when induced by infection or through vaccination. Using a mouse model, we found that prior HCoV-OC43 S-targeted immunity primes neutralizing antibody responses to otherwise subimmunogenic SARS-CoV-2 S exposure and promotes S2-targeting antibody responses. Moreover, vaccination with SARS-CoV-2 S2 elicited antibodies in mice that neutralized diverse animal and human alphacoronaviruses and betacoronaviruses in vitro and provided a degree of protection against SARS-CoV-2 challenge in vivo. Last, in mice with a history of SARS-CoV-2 Wuhan-based S vaccination, further S2 vaccination induced broader neutralizing antibody response than booster Wuhan S vaccination, suggesting that it may prevent repertoire focusing caused by repeated homologous vaccination. These data establish the protective value of an S2-targeting vaccine and support the notion that S2 vaccination may better prepare the immune system to respond to the changing nature of the S1 subunit in SARS-CoV-2 variants of concern, as well as to future coronavirus zoonoses.
The intasome is the basic recombination unit of retroviral integration, comprising the integrase protein and the ends of the viral DNA made by reverse transcription. Clinical inhibitors ...preferentially target the DNA-bound form of integrase as compared with the free protein, highlighting the critical requirement for detailed understanding of HIV-1 intasome structure and function. Although previous biochemical studies identified integrase residues that contact the DNA, structural details of protein–protein and protein–DNA interactions within the functional intasome were lacking. The recent crystal structure of the prototype foamy virus (PFV) integrase—viral DNA complex revealed numerous details of this related integration machine. Structures of drug-bound PFV intasomes moreover elucidated the mechanism of inhibitor action. Herein we present a model for the HIV-1 intasome assembled using the PFV structure as template. Our results pinpoint previously identified protein–DNA contacts within the quaternary structure and reveal hitherto unknown roles for Arg20 and Lys266 in DNA binding and integrase function. Models for clinical inhibitors bound at the HIV-1 integrase active site were also constructed and compared with previous studies. Our findings highlight the structural basis for HIV-1 integration and define the mechanism of its inhibition, which should help in formulating new drugs to inhibit viruses resistant to first-in-class compounds.
There is an urgent need to understand the nature of immune responses against SARS-CoV-2, to inform risk-mitigation strategies for people living with HIV (PLWH). Here we show that the majority of PLWH ...with ART suppressed HIV viral load, mount a detectable adaptive immune response to SARS-CoV-2. Humoral and SARS-CoV-2-specific T cell responses are comparable between HIV-positive and negative subjects and persist 5-7 months following predominately mild COVID-19 disease. T cell responses against Spike, Membrane and Nucleoprotein are the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. We further show that the overall magnitude of SARS-CoV-2-specific T cell responses relates to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH. These findings suggest that inadequate immune reconstitution on ART, could hinder immune responses to SARS-CoV-2 with implications for the individual management and vaccine effectiveness in PLWH.
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