The causative agent of the COVID-19 pandemic, SARS-CoV-2, is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the ACE2 receptor and is cleaved by TMPRSS2. However, whether S mutations affect SARS-CoV-2 cell entry remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than that of SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural and binding analyses. Nevertheless, the D614G mutation does not affect neutralization by antisera against prototypic viruses. Taken together, we conclude that the D614G mutation increases cell entry by acquiring higher affinity to ACE2 while maintaining neutralization susceptibility. Based on these findings, further worldwide surveillance is required to understand SARS-CoV-2 transmissibility among humans.

APOBEC3G (A3G), a host protein that inhibits HIV-1 reverse transcription and replication in the absence of Vif, displays cytidine deaminase and single-stranded (ss) nucleic acid binding activities. HIV-1 nucleocapsid protein (NC) also binds nucleic acids and has a unique property, nucleic acid chaperone activity, which is crucial for efficient reverse transcription. Here we report the interplay between A3G, NC and reverse transcriptase (RT) and the effect of highly purified A3G on individual reactions that occur during reverse transcription. We find that A3G did not affect the kinetics of NC-mediated annealing reactions, nor did it inhibit RNase H cleavage. In sharp contrast, A3G significantly inhibited all RT-catalyzed DNA elongation reactions with or without NC. In the case of ( − ) strong-stop DNA synthesis, the inhibition was independent of A3G's catalytic activity. Fluorescence anisotropy and single molecule DNA stretching analyses indicated that NC has a higher nucleic acid binding affinity than A3G, but more importantly, displays faster association/disassociation kinetics. RT binds to ssDNA with a much lower affinity than either NC or A3G. These data support a novel mechanism for deaminase-independent inhibition of reverse transcription that is determined by critical differences in the nucleic acid binding properties of A3G, NC and RT.

APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits human immunodeficiency virus type 1 replication in the absence of Vif. Here, we provide a comprehensive molecular analysis of the deaminase and nucleic acid binding activities of human APO3G using a pure system containing only one protein component, i.e., highly purified, catalytically active enzyme expressed in a baculovirus system. We demonstrate that APO3G deaminates cytosines in single-stranded DNA (ssDNA) only, whereas it binds efficiently to ssDNA and ssRNA, about half as well to a DNA/RNA hybrid, and poorly to double-stranded DNA and RNA. In addition, the base specificities for deamination and binding of ssDNA are not correlated. The minimum length required for detection of APO3G binding to an ssDNA oligonucleotide in an electrophoretic mobility shift assay is 16 nucleotides. Interestingly, if nucleocapsid protein and APO3G are present in the same reaction, we find that they do not interfere with each other's binding to RNA and a complex containing the RNA and both proteins is formed. Finally, we also identify the functional activities of each zinc finger domain. Thus, although both zinc finger domains have the ability to bind nucleic acids, the first zinc finger contributes more to binding and APO3G encapsidation into virions than finger two. In contrast, deamination is associated exclusively with the second zinc finger. Moreover, zinc finger two is more important than finger one for the antiviral effect, demonstrating a correlation between deaminase and antiviral activities.

In HIV-1-infected patients, antiretroviral therapy (ART) is a key factor that may impact commensal microbiota and cause the emergence of side effects. However, it is not fully understood how long-term ART regimens have diverse impacts on the microbial compositions over time. Here, we performed 16S ribosomal RNA gene sequencing of the fecal and salivary microbiomes in patients under different long-term ART. We found that ART, especially conventional nucleotide/nucleoside reverse transcriptase inhibitor (NRTI)-based ART, has remarkable impacts on fecal microbial diversity: decreased α-diversity and increased ß-diversity over time. In contrast, dynamic diversity changes in the salivary microbiome were not observed. Comparative analysis of bacterial genus compositions showed a propensity for Prevotella-enriched and Bacteroides-poor gut microbiotas in patients with ART over time. In addition, we observed a gradual reduction in Bacteroides but drastic increases in Succinivibrio and/or Megasphaera under conventional ART. These results suggest that ART, especially NRTI-based ART, has more suppressive impacts on microbiota composition and diversity in the gut than in the mouth, which potentially causes intestinal dysbiosis in patients. Therefore, NRTI-sparing ART, especially integrase strand transfer inhibitor (INSTI)- and/or non-nucleotide reverse transcriptase inhibitor (NNRTI)-containing regimens, might alleviate the burden of intestinal dysbiosis in HIV-1-infected patients under long-term ART.

APOBEC3G (APO3G) is a cytidine deaminase that restricts replication of vif -defective human immunodeficiency virus type 1 (HIV-1). Like other members of the cellular deaminase family, APO3G has the propensity to form homo-multimers. In the current study, we investigated the functional determinants for multimerization of human APO3G and studied the role of APO3G multimerization for catalytic activity, virus encapsidation, and antiviral activity. We found that human APO3G is capable of forming multimeric complexes in transfected HeLa cells. Interestingly, multimerization of APO3G was exquisitely sensitive to RNase treatment, suggesting that interaction of APO3G subunits is facilitated or stabilized by an RNA bridge. Mutation of a conserved cysteine residue (C97) that is part of an N-terminal zinc-finger motif in APO3G abolished multimerization of APO3G; however, the C97 mutation inhibited neither in vitro deaminase activity nor antiviral function of APO3G. These results suggest that monomeric APO3G is both catalytically active and has antiviral activity. Interference studies employing either catalytically inactive or packaging-incompetent APO3G variants suggest that wild-type APO3G is packaged into HIV-1 particles in monomeric form. These results provide novel insights into the catalytic function and antiviral property of APO3G and demonstrate an important role for C97 in the RNA-dependent multimerization of this protein.