There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2′-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m⁷GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m⁷GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.

The Repeats-in-Toxins (RTX) family of proteins classically consists of cytolysins and hemolysins. Over the past decade, genome sequencing revealed the existence of very large members of this family. These are all repetitive proteins ranging in size from 200 to 900 kDa that function as toxins or adhesins. Many are exported by Type I secretion. One major new subfamily is the large repetitive RTX adhesins and biofilm-associated proteins. These are characterized by 80- to 300-amino-acid repeats ordered in tandem, although the sequence and number of the repeats vary by protein. The second major new subfamily is the multifunctional-autoprocessing RTX toxins, which are associated with cytotoxicity and pathogenesis. These proteins are in turn distantly related to Yersinia hypothetical RTX proteins that may autoprocess by a similar mechanism. This review discusses current knowledge regarding the structure and function of these new subfamilies of RTX proteins.

The Gram-negative pathogen Vibrio cholerae causes diarrheal disease through the export of enterotoxins. The V. cholerae RTX toxin was previously identified and characterized by its ability to round human laryngeal epithelial (HEp-2) cells. Further investigation determined that cell rounding is caused by the depolymerization of actin stress fibers, through the unique mechanism of covalent actin cross-linking. In this study, we identify a domain within the full-length RTX toxin that is capable of mediating the cross-linking reaction when transiently expressed within eukaryotic cells. A structure/function analysis of the actin cross-linking domain (ACD) reveals that a 412-aa, or a 47.8-kDa, region is essential for cross-linking activity. When this domain is deleted from the full-length toxin gene, actin cross-linking, but not cell rounding, is eliminated, indicating that this toxin carries multiple dissociable activities. The ACD shares 59% amino acid identity with a hypothetical protein from V. cholerae , VC1416, and transient expression of the C-terminal domain of VC1416 also results in actin cross-linking in eukaryotic cells. The presence of this second ACD linked to an Rhs -like element suggests that V. cholerae acquired the domain by horizontal gene transfer and the ACD was inserted into the RTX toxin by gene duplication through the evolution of V. cholerae .

Vibrio vulnificus is a pathogen that causes both severe necrotizing wound infections and life-threatening food-borne infections. Food-borne infection is particularly lethal as the infection can progress rapidly to primary septicemia resulting in death from septic shock and multiorgan failure. In this study, we use both bioluminescence whole animal imaging and V. vulnificus bacterial colonization of orally infected mice to demonstrate that the secreted multifunctional-autoprocessing RTX toxin (MARTX_Vv) and the cytolysin/hemolysin VvhA of clinical isolate CMCP6 have an important function in the gut to promote early in vivo growth and dissemination of this pathogen from the small intestine to other organs. Using histopathology, we find that both cytotoxins can cause villi disruption, epithelial necrosis, and inflammation in the mouse small intestine. A double mutant deleted of genes for both cytotoxins was essentially avirulent, did not cause intestinal epithelial tissue damage, and was cleared from infected mice by 36 hours by an effective immune response. Therefore, MARTX_Vv and VvhA seem to play an additive role for pathogenesis of CMCP6 causing intestinal tissue damage and inflammation that then promotes dissemination of the infecting bacteria to the bloodstream and other organs. In the absence of these two secreted factors, we propose that this bacterium is unable to cause intestinal infection in humans. Vibrio vulnificus causes disease both by infection of wounds from seawater and by consumption of contaminated foods, especially oysters. Wound infection results in necrotizing fasciitis and edema in extremities with mortality of ∼25% as the incidence of septicemia is low. Contaminated food consumption by contrast can lead to highly invasive infections that progress rapidly from an intestinal infection to primary septicemia. Case-fatality rates are ≥50%, with rates as high as 100% in individuals who receive no antibiotic therapy. The aim of this study is to elucidate virulence mechanisms of food-borne infection of the most highly virulent strains of V. vulnificus. We developed a novel intragastric infection model for a highly virulent clinical isolate from Korea in which we can observe the bacterial load in live mice and applied this to study of wild type and strains genetically altered to delete genes for two secreted cytotoxins. Using this model, we show that both the multifunctional-autoprocessing RTX toxin (MARTX_Vv) and the cytolysin VvhA contribute to rapid in vivo growth of bacteria and that the presence of these factors directly correlates with mouse mortality. These exotoxins are then directly linked to intestinal damage and inflammation.

The repeats-in-toxin (RTX) exoproteins are a diverse col- lection of proteins exported via type I secretion by gram-neg- ative bacteria. These proteins include the pore-forming RTX toxins typified by -hemolysin of Escherichia coli and Bordetella pertussis adenylate cyclase toxin (ACT) and also secreted en- zymes such as metalloproteases and lipases, S-layer proteins, a nodulation signaling factor, and the Fe-regulated FrpC "clip- and-link" toxin of Neisseria meningitidis (24, 27). In 1999, a new toxin from Vibrio cholerae was discovered that was initially categorized in the RTX toxin family and simply named "the RTX toxin" or VcRtxA (22). This toxin is pro- duced by nearly all clinical and environmental isolates of V. cholerae, including the El Tor O1 strains responsible for the current cholera pandemic plus a broad selection of O139 and non-O1/non-O139 strains (7, 9, 10, 13). The toxin has been associated with increased epithelial cell damage in a mouse lung infection model (14) and has also been associated with increased virulence in a mouse gut infection model, although its role in intestinal disease is currently unclear (26). Recently, a related toxin from Vibrio vulnificus has been described and found to be important for virulence in mouse models (21, 23). On-going study of the mechanism of action of the V. cholerae toxin in vitro has revealed that it is a totally novel type of toxin with many features that distinguish it from other RTX toxins. In addition, release of genomic sequences from human, ma- rine, and insect pathogens has indicated that the toxins from V. cholerae and V. vulnificus are not unique but rather are the first characterized members of a new family of the RTX exopro- teins. Within this new family, the structural properties of the toxins are highly conserved, but the catalytic activities carried are predicted to vary since the toxins are assembled as mosaics of 10 activity domains assorted among the various toxins. In this review, the structure and function of the V. cholerae toxin are discussed in detail, revealing that this is a novel toxin distinct from the other RTX toxins. The structural features that define the new family and the mosaic structure of the activity domains are also discussed. Overall, it is shown that the V. cholerae toxin is a multifunctional autoprocessing RTX toxin, renamed MARTXVc, that represents a larger group of MARTX toxins produced by at least eight gram-negative spe- cies. NOVEL REPEAT STRUCTURE OF MARTX TOXINS A common property of all RTX pore-forming toxins is their large size; their molecular masses range from 100 to 177 kDa (24). At the C terminus of these large proteins are "GD-rich" nonapeptide repeats (GGXGXDX(L/I/V/W/Y/F)X), where X is any amino acid), which are the defining characteristic of the RTX exoproteins (20). These repeats have been shown to fold to form a stable -roll structure that binds Ca 2 (3) and are thought to be involved in proper insertion of the toxins into the eukaryotic cytoplasmic membrane (29).

Vibrio vulnificus is a food-borne bacterial pathogen associated with 1% of all food-related deaths, predominantly because of consumption of contaminated seafood. The ability of V. vulnificus to cause disease is linked to the production of a large cytotoxin called the “multifunctional-autoprocessing RTX” (MARTX _Vv ) toxin, a factor shown here to be an important virulence factor by the intragastric route of infection in mice. In this study, we examined genetic variation of the rtxA1 gene that encodes MARTX _Vv in 40 V. vulnificus Biotype 1 strains and found four distinct variants of rtxA1 that encode toxins with different arrangements of effector domains. We provide evidence that these variants arose by recombination either with rtxA genes carried on plasmids or with the rtxA gene of Vibrio anguillarum . Contrary to expected results, the most common rtxA1 gene variant in clinical-type V. vulnificus encodes a toxin with reduced potency and is distinct from the toxin produced by strains isolated from market oysters. These results indicate that an important virulence factor of V. vulnificus is undergoing significant genetic rearrangement and may be subject to selection for reduced virulence in the environment. This finding would imply further that in the future on-going genetic variation of the MARTX _Vv toxins could result in the emergence of novel strains with altered virulence in humans.