The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact, and that the conformation is shifted towards an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.

AbstractVirus genome sequence variants that appear over the course of an outbreak can be exploited to map the trajectory of the virus from one susceptible host to another. While such variants are usually of no functional significance, in some cases they may allow the virus to transmit faster, change disease severity, or confer resistance to antiviral therapies. Since the discovery of SARS-CoV-2 as the cause of COVID-19, the virus has spread around the globe, and thousands of SARS-CoV-2 genomes have been sequenced. The rate of sequence variation among SARS-CoV-2 isolates is modest for an RNA virus but the enormous number of human-to-human transmission events has provided abundant opportunity for selection of sequence variants. Among these, the SARS-CoV-2 Spike protein variant, D614G, was not present in the presumptive common ancestor of this zoonotic virus but was first detected in late January in Germany and China. The D614G variant steadily increased in frequency and now constitutes >97% of isolates world-wide, raising the question whether D614G confers a replication advantage to SARS-CoV-2. Structural models predict that D614G would disrupt contacts between the S1 and S2 domains of the Spike protein and cause significant shifts in conformation. Using single-cycle vectors we showed that D614G is three to nine-fold more infectious than the ancestral form on human lung and colon cell lines, as well as on other human cell lines rendered permissive by ectopic expression of human ACE2 and TMPRSS2, or by ACE2 orthologues from pangolin, pig, dog, or cat. Nonetheless, monoclonal antibodies targeting the receptor binding domain of the SARS-CoV-2 Spike protein retain full neutralization potency. These results suggest that D614G was selected for increased human-to-human transmission, that it contributed to the rapidity of SARS-CoV-2 spread around the world, and that it does not confer resistance to antiviral therapies targeting the receptor binding domain.

Host factors that silence provirus transcription in CD4 + memory T cells help HIV-1 escape eradication by the host immune system and by antiviral drugs 1 . These same factors, though, must be overcome for HIV-1 to propagate. Here we show that Vpx and Vpr encoded by diverse primate immunodeficiency viruses activate provirus transcription. Vpx and Vpr are adaptor proteins for the DCAF1-CUL4A/B E3 ubiquitin ligase that degrade SAMHD1 and increase reverse transcription 2 – 4 . Nonetheless, Vpx and Vpr have effects on reporter gene expression that are not explained by SAMHD1 degradation 5 – 8 . A screen for factors that mimic these effects identified the Human Silencing Hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN), and MORC2 9 – 13 . Vpx associated with the HUSH complex and decreased steady-state level of these proteins in a DCAF1/CUL4A/B/proteasome-dependent manner 14 , 15 . Replication kinetics of HIV-1 and SIV MAC was accelerated to a similar extent by vpx or FAM208A knockdown. Finally, vpx increased steady-state levels of LINE-1 ORF1p, as previously described for FAM208A disruption 11 . These results demonstrate that the HUSH complex represses primate immunodeficiency virus transcription, and that, to counteract this restriction, viral Vpx or Vpr proteins degrade the HUSH complex.

Including: Extended Data Figures 1, 2, 3 and 4 with corresponding Figure Legends. Supplementary Tables 1, 2 and 3 Extended Data Figure 1. Gating strategy for flow cytometry experiments assessing single cycle infectivity.Macrophage and dendritic cell populations, previously enriched as per the methods, were gated by SSC-A vs. FSC-A, as indicated, and then the GFP + population was plotted vs. FSC-A. Enriched CD4 + T cells were gated by SSC-A vs FSC-A, as indicated, then a singlet population was gated from FSC-H vs. FSC-A, and finally GFP + cells were plotted vs. FSC-A.

SUMMARY B lymphocyte hyperactivation and hypergammaglobulinemia are pathogenic manifestations of HIV-1 infection. Here we provide evidence that these B cell defects are driven by factors produced by HIV-1 infected macrophages and that Nef is necessary for this activity. In vitro, HIV-1 infected macrophages or macrophages expressing Nef promoted B cell activation and differentiation to immunoglobulin-secreting cells. Activation of NF-κB by Nef induced secretion of the acute-phase reactant ferritin and ferritin was necessary and sufficient for these B cell effects. The extent of hypergammaglobulinemia in HIV-1 infected individuals correlated directly with plasma ferritin levels and with viral load. We further demonstrate that induction of ferritin and hypergammaglobulinemia could be recapitulated when Nef was specifically expressed in macrophages and T cells of transgenic mice. Collectively, these results reveal the presence of a pathogenic determinant within the Nef protein of HIV-1 which governs B cell defects in HIV-1 infection.