Tetherin, an interferon-inducible membrane protein, inhibits the release of nascent enveloped viral particles from the surface of infected cells. However, the mechanisms underlying virion retention ...have not yet been fully delineated. Here, we employ biochemical assays and engineered tetherin proteins to demonstrate conclusively that virion tethers are composed of the tetherin protein itself, and to elucidate the configuration and topology that tetherin adopts during virion entrapment. We demonstrate that tetherin dimers adopt an "axial" configuration, in which pairs of transmembrane domains or pairs of glycosylphosphatidyl inositol anchors are inserted into assembling virion particles, while the remaining pair of membrane anchors remains embedded in the infected cell membrane. We use quantitative western blotting to determine that a few dozen tetherin dimers are used to tether each virion particle, and that there is ∼3- to 5-fold preference for the insertion of glycosylphosphatidyl inositol anchors rather than transmembrane domains into tethered virions. Cumulatively, these results demonstrate that axially configured tetherin homodimers are directly responsible for trapping virions at the cell surface. We suggest that insertion of glycosylphosphatidyl inositol anchors may be preferred so that effector functions that require exposure of the tetherin N-terminus to the cytoplasm of infected cells are retained.
HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable ...understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells.
Viral infections are often detrimental to host survival and reproduction. Consequently, hosts have evolved a variety of mechanisms to defend themselves against viruses. A component of this arsenal is ...a set of proteins, termed restriction factors, which exhibit direct antiviral activity. Among these are several classes of proteins (APOBEC3, TRIM5, Tetherin, and SAMHD1) that inhibit the replication of human and simian immunodeficiency viruses. Here, we outline the features, mechanisms, and evolution of these defense mechanisms. We also speculate on how restriction factors arose, how they might interact with the conventional innate and adaptive immune systems, and how an understanding of these intrinsic cellular defenses might be usefully exploited.
The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific ...antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.
Retroviruses have long been a fertile model for discovering host-pathogen interactions and their associated biological principles and processes. These advances have not only informed fundamental ...concepts of viral replication and pathogenesis but have also provided novel insights into host cell biology. This is illustrated by the recent descriptions of host-encoded restriction factors that can serve as effective inhibitors of retroviral replication. Here, we review our understanding of the three restriction factors that have been widely shown to be potent inhibitors of HIV-1: namely, APOBEC3G, TRIM5α, and tetherin. In each case, we discuss how these unrelated proteins were identified, the mechanisms by which they inhibit replication, the means used by HIV-1 to evade their action, and their potential contributions to viral pathogenesis as well as inter- and intraspecies transmission.
Abstract
Background
Understanding the longitudinal trajectory of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is crucial for diagnosis of prior infection and predicting ...future immunity.
Methods
We conducted a longitudinal analysis of coronavirus disease 2019 convalescent patients, with neutralizing antibody assays and SARS-CoV-2 serological assay platforms using SARS-CoV-2 spike (S) or nucleocapsid (N) antigens.
Results
Sensitivities of serological assays in diagnosing prior SARS-CoV-2 infection changed with time. One widely used commercial platform that had an initial sensitivity of >95% declined to 71% at 81–100 days after diagnosis. The trajectories of median binding antibody titers measured over approximately 3–4 months were not dependent on the use of SARS-CoV-2 N or S proteins as antigen. The median neutralization titer decreased by approximately 45% per month. Each serological assay gave quantitative antibody titers that were correlated with SARS-CoV-2 neutralization titers, but S-based serological assay measurements better predicted neutralization potency. Correlation between S-binding and neutralization titers deteriorated with time, and decreases in neutralization titers were not predicted by changes in S-binding antibody titers.
Conclusions
Different SARS-CoV-2 serological assays are more or less well suited for surveillance versus prediction of serum neutralization potency. Extended follow-up should facilitate the establishment of appropriate serological correlates of protection against SARS-CoV-2 reinfection.
Analysis of coronavirus disease 2019 convalescent patients reveals that neutralizing antibody levels decline rapidly early after infection. Some clinical serological assay platforms give quantitative outputs that predict neutralizing antibody titer, but in some diagnostic sensitivity deteriorates with time after infection.
More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite ...the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies
. Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines
. In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals
. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern
. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.
The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in individuals who are SARS-CoV-2 convalescent and vaccinated are key determinants of neutralization breadth ...and the genetic barrier to viral escape
. Using HIV-1 pseudotypes and plasma selection experiments with vesicular stomatitis virus/SARS-CoV-2 chimaeras
, here we show that multiple neutralizing epitopes, within and outside the receptor-binding domain, are variably targeted by human polyclonal antibodies. Antibody targets coincide with spike sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic 'polymutant' spike protein pseudotypes that resisted polyclonal antibody neutralization to a similar degree as circulating variants of concern. By aggregating variant of concern-associated and antibody-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in the SARS-CoV-2 spike protein are sufficient to generate pseudotypes with near-complete resistance to the polyclonal neutralizing antibodies generated by individuals who are convalescent or recipients who received an mRNA vaccine. However, plasma from individuals who had been infected and subsequently received mRNA vaccination neutralized pseudotypes bearing this highly resistant SARS-CoV-2 polymutant spike, or diverse sarbecovirus spike proteins. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against potential future sarbecovirus pandemics.
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