Potent neutralization by a receptor binding domain monoclonal antibody with broad specificity for SARS-CoV-2 JN.1 and other variants.

SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. While increasing herd immunity, current vaccines, and therapeutics have improved outcomes for some; prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) - ACE2 fusion protein and differential selection process with native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan and more recent Omicron JN.1 strain, as well as SARS-CoV. Structure determination of the SARS-CoV-2 EG5.1 Spike/1301B7 Fab complex by cryo-electron microscopy at 3.1Å resolution demonstrates 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain, making contacts using CDRs1-3, as well as framework region 3 (FR3). Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.

LIF/JAK2/STAT1 Signaling Enhances Production of Galactose-Deficient IgA1 by IgA1-Producing Cell Lines Derived From Tonsils of Patients With IgA Nephropathy.

Introduction: Galactose-deficient IgA1 (Gd-IgA1) plays a key role in the pathogenesis of IgA nephropathy (IgAN). Tonsillectomy has been beneficial to some patients with IgAN, possibly due to the removal of tonsillar cytokine-activated cells producing Gd-IgA1. To test this hypothesis, we used immortalized IgA1-producing cell lines derived from tonsils of patients with IgAN or obstructive sleep apnea (OSA) and assessed the effect of leukemia inhibitory factor (LIF) or oncostatin M (OSM) on Gd-IgA1 production. Methods: Gd-IgA1 production was measured by lectin enzyme-linked immunosorbent assay; JAK-STAT signaling in cultured cells was assessed by immunoblotting of cell lysates; and validated by using small interfering RNA (siRNA) knock-down and small-molecule inhibitors. Results: IgAN-derived cells produced more Gd-IgA1 than the cells from patients with OSA, and exhibited elevated Gd-IgA1 production in response to LIF, but not OSM. This effect was associated with dysregulated STAT1 phosphorylation, as confirmed by STAT1 siRNA knock-down. JAK2 inhibitor, AZD1480 exhibited a dose-dependent inhibition of the LIF-induced Gd-IgA1 overproduction. Unexpectedly, high concentrations of AZD1480, but only in the presence of LIF, reduced Gd-IgA1 production in the cells derived from patients with IgAN to that of the control cells from patients with OSA. Based on modeling LIF-LIFR-gp130-JAK2 receptor complex, we postulate that LIF binding to LIFR may sequester gp130 and/or JAK2 from other pathways; and when combined with JAK2 inhibition, enables full blockade of the aberrant O-glycosylation pathways in IgAN. Conclusion: In summary, IgAN cells exhibit LIF-mediated overproduction of Gd-IgA1 due to abnormal signaling. JAK2 inhibitors can counter these LIF-induced effects and block Gd-IgA1 synthesis in IgAN.

Current Understanding of Complement Proteins as Therapeutic Targets for the Treatment of Immunoglobulin A Nephropathy.

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide and a frequent cause of kidney failure. Currently, the diagnosis necessitates a kidney biopsy, with routine immunofluorescence microscopy revealing IgA as the dominant or co-dominant immunoglobulin in the glomerular immuno-deposits, often with IgG and sometimes IgM or both. Complement protein C3 is observed in most cases. IgAN leads to kidney failure in 20-40% of patients within 20 years of diagnosis and reduces average life expectancy by about 10 years. There is increasing clinical, biochemical, and genetic evidence that the complement system plays a paramount role in the pathogenesis of IgAN. The presence of C3 in the kidney immuno-deposits differentiates the diagnosis of IgAN from subclinical glomerular mesangial IgA deposition. Markers of complement activation via the lectin and alternative pathways in kidney-biopsy specimens are associated with disease activity and are predictive of poor outcome. Levels of select complement proteins in the circulation have also been assessed in patients with IgAN and found to be of prognostic value. Ongoing genetic studies have identified at least 30 loci associated with IgAN. Genes within some of these loci encode complement-system regulating proteins that can interact with immune complexes. The growing appreciation for the central role of complement components in IgAN pathogenesis highlighted these pathways as potential treatment targets and sparked great interest in pharmacological agents targeting the complement cascade for the treatment of IgAN, as evidenced by the plethora of ongoing clinical trials.

Discontinuous L-binding motifs in the transactivation domain of the vesicular stomatitis virus P protein are required for terminal de novo transcription initiation by the L protein.

The phospho- (P) protein, the co-factor of the RNA polymerase large (L) protein, of vesicular stomatitis virus (VSV, a prototype of nonsegmented negative-strand RNA viruses) plays pivotal roles in transcription and replication. However, the precise mechanism underlying the transcriptional transactivation by the P protein has remained elusive. Here, using an in vitro transcription system and a series of deletion mutants of the P protein, we mapped a region encompassing residues 51-104 as a transactivation domain (TAD) that is critical for terminal de novo initiation, the initial step of synthesis of the leader RNA and anti-genome/genome, with the L protein. Site-directed mutagenesis revealed that conserved amino acid residues in three discontinuous L-binding sites within the TAD are essential for the transactivation activity of the P protein or important for maintaining its full activity. Importantly, relative inhibitory effects of TAD point mutations on synthesis of the full-length leader RNA and mRNAs from the 3'-terminal leader region and internal genes, respectively, of the genome were similar to those on terminal de novo initiation. Furthermore, any of the examined TAD mutations did not alter the gradient pattern of mRNAs synthesized from internal genes, nor did they induce the production of readthrough transcripts. These results suggest that these TAD mutations impact mainly terminal de novo initiation but rarely other steps (e.g., elongation, termination, internal initiation) of single-entry stop-start transcription. Consistently, the mutations of the essential or important amino acid residues within the P TAD were lethal or deleterious to VSV replication in host cells. IMPORTANCE RNA-dependent RNA polymerase L proteins of nonsegmented negative-strand RNA viruses belonging to the Mononegavirales order require their cognate co-factor P proteins or their counterparts for genome transcription and replication. However, exact roles of these co-factor proteins in modulating functions of L proteins during transcription and replication remain unknown. In this study, we revealed that three discrete L-binding motifs within a transactivation domain of the P protein of vesicular stomatitis virus, a prototypic nonsegmented negative-strand RNA virus, are required for terminal de novo initiation mediated by the L protein, which is the first step of synthesis of the leader RNA as well as genome/anti-genome.

B cell and antibody targeting of transplant alloantigen epitopes is supported by both self and allo-recognition.

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific (alloreactive) B cell response in kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We identified 14 distinct alloreactive B cell lineages, which spanned the rejected organ and blood and expressed high-affinity anti-donor HLA-specific B cell receptors, many of which were clonally linked to circulating DSA. The alloreactive B cell response was focused on exposed, solvent-accessible mismatched HLA residues, while also demonstrating extensive contacts with self-HLA residues. Consistent with structural evidence of self-recognition, measurable self-reactivity by donor-specific B cells was common and positively correlated with anti-donor affinity maturation. Thus, allo- and self-reactive signatures appeared to converge, suggesting that during AMR, the recognition of non-self and breaches of tolerance conspire to produce a pathogenic donor-specific adaptive response.

MDA5-dependent responses contribute to autoimmune diabetes progression and hindrance.

Type 1 diabetes (T1D) is an autoimmune disease resulting in pancreatic ß cell destruction. Coxsackievirus B3 (CVB3) infection and melanoma differentiation-associated protein 5-dependent (MDA5-dependent) antiviral responses are linked with T1D development. Mutations within IFIH1, coding for MDA5, are correlated with T1D susceptibility, but how these mutations contribute to T1D remains unclear. Utilizing nonobese diabetic (NOD) mice lacking Ifih1 expression (KO) or containing an in-frame deletion within the ATPase site of the helicase 1 domain of MDA5 (ΔHel1), we tested the hypothesis that partial or complete loss-of-function mutations in MDA5 would delay T1D by impairing proinflammatory pancreatic macrophage and T cell responses. Spontaneous T1D developed in female NOD and KO mice similarly, but was significantly delayed in ΔHel1 mice, which may be partly due to a concomitant increase in myeloid-derived suppressor cells. Interestingly, KO male mice had increased spontaneous T1D compared with NOD mice. Whereas NOD and KO mice developed CVB3-accelerated T1D, ΔHel1 mice were protected partly due to decreased type I IFNs, pancreatic infiltrating TNF+ macrophages, IFN-γ+CD4+ T cells, and perforin+CD8+ T cells. Furthermore, ΔHel1 MDA5 protein had reduced ATP hydrolysis compared with wild-type MDA5. Our results suggest that dampened MDA5 function delays T1D, yet loss of MDA5 promotes T1D.

Cytokines and Production of Aberrantly O-Glycosylated IgA1, the Main Autoantigen in IgA Nephropathy.

Immunoglobulin A (IgA) nephropathy is the most common primary glomerulonephritis worldwide, with no disease-specific treatment and up to 40% of patients progressing to kidney failure. IgA nephropathy (IgAN), characterized by IgA1-containing immunodeposits in the glomeruli, is considered to be an autoimmune disease in which the kidneys are injured as innocent bystanders. Glomerular immunodeposits are thought to originate from the circulating immune complexes that contain aberrantly O-glycosylated IgA1, the main autoantigen in IgAN, bound by IgG autoantibodies. A common clinical manifestation associated with IgAN includes synpharyngitic hematuria at disease onset or during disease activity. This observation suggests a connection of disease pathogenesis with an activated mucosal immune system of the upper-respiratory and/or gastrointestinal tract and IgA1 glycosylation. In fact, some cytokines can enhance production of aberrantly O-glycosylated IgA1. This process involves abnormal cytokine signaling in IgA1-producing cells from patients with IgAN. In this article, we present our view of pathogenesis of IgAN and review how some cytokines can contribute to the disease process by enhancing production of aberrantly glycosylated IgA1. We also review current clinical trials of IgAN based on cytokine-targeting therapeutic approaches.

Atomic view of the HIV-1 matrix lattice; implications on virus assembly and envelope incorporation.

During the late phase of HIV type 1 (HIV-1) infection cycle, the virally encoded Gag polyproteins are targeted to the inner leaflet of the plasma membrane (PM) for assembly, formation of immature particles, and virus release. Gag binding to the PM is mediated by interactions of the N-terminally myristoylated matrix (myrMA) domain with phosphatidylinositol 4,5-bisphosphate. Formation of a myrMA lattice on the PM is an obligatory step for the assembly of immature HIV-1 particles and envelope (Env) incorporation. Atomic details of the myrMA lattice and how it mediates Env incorporation are lacking. Herein, we present the X-ray structure of myrMA at 2.15 Å. The myrMA lattice is arranged as a hexamer of trimers with a central hole, thought to accommodate the C-terminal tail of Env to promote incorporation into virions. The trimer­trimer interactions in the lattice are mediated by the N-terminal loop of one myrMA molecule and α-helices I­II, as well as the 310 helix of a myrMA molecule from an adjacent trimer. We provide evidence that substitution of MA residues Leu13 and Leu31, previously shown to have adverse effects on Env incorporation, induced a conformational change in myrMA, which may destabilize the trimer­trimer interactions within the lattice. We also show that PI(4,5)P2 is capable of binding to alternating sites on MA, consistent with an MA­membrane binding mechanism during assembly of the immature particle and upon maturation. Altogether, these findings advance our understanding of a key mechanism in HIV-1 particle assembly.

GDP polyribonucleotidyltransferase domain of vesicular stomatitis virus polymerase regulates leader-promoter escape and polyadenylation-coupled termination during stop-start transcription.

The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain of the vesicular stomatitis virus (VSV) L protein possesses a dual-functional "priming-capping loop" that governs terminal de novo initiation for leader RNA synthesis and capping of monocistronic mRNAs during the unique stop-start transcription cycle. Here, we investigated the roles of basic amino acid residues on a helix structure directly connected to the priming-capping loop in viral RNA synthesis and identified single point mutations that cause previously unreported defective phenotypes at different steps of stop-start transcription. Mutations of residue R1183 (R1183A and R1183K) dramatically reduced the leader RNA synthesis activity by hampering early elongation, but not terminal de novo initiation or productive elongation, suggesting that the mutations negatively affect escape from the leader promoter. On the other hand, mutations of residue R1178 (R1178A and R1178K) decreased the efficiency of polyadenylation-coupled termination of mRNA synthesis at the gene junctions, but not termination of leader RNA synthesis at the leader-to-N-gene junction, resulting in the generation of larger amounts of aberrant polycistronic mRNAs. In contrast, both the R1183 and R1178 residues are not essential for cap-forming activities. The R1183K mutation was lethal to VSV, whereas the R1178K mutation attenuated VSV and triggered the production of the polycistronic mRNAs in infected cells. These observations suggest that the PRNTase domain plays multiple roles in conducting accurate stop-start transcription beyond its known role in pre-mRNA capping.

Pathogenesis of IgA Nephropathy: Current Understanding and Implications for Development of Disease-Specific Treatment.

IgA nephropathy, initially described in 1968 as a kidney disease with glomerular "intercapillary deposits of IgA-IgG", has no disease-specific treatment and is a common cause of kidney failure. Clinical observations and laboratory analyses suggest that IgA nephropathy is an autoimmune disease wherein the kidneys are damaged as innocent bystanders due to deposition of IgA1-IgG immune complexes from the circulation. A multi-hit hypothesis for the pathogenesis of IgA nephropathy describes four sequential steps in disease development. Specifically, patients with IgA nephropathy have elevated circulating levels of IgA1 with some O-glycans deficient in galactose (galactose-deficient IgA1) and these IgA1 glycoforms are recognized as autoantigens by unique IgG autoantibodies, resulting in formation of circulating immune complexes, some of which deposit in glomeruli and activate mesangial cells to induce kidney injury. This proposed mechanism is supported by observations that (i) glomerular immunodeposits in patients with IgA nephropathy are enriched for galactose-deficient IgA1 glycoforms and the corresponding IgG autoantibodies; (ii) circulatory levels of galactose-deficient IgA1 and IgG autoantibodies predict disease progression; and (iii) pathogenic potential of galactose-deficient IgA1 and IgG autoantibodies was demonstrated in vivo. Thus, a better understanding of the structure-function of these immunoglobulins as autoantibodies and autoantigens will enable development of disease-specific treatments.

Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Natural and Recombinant SARS-CoV-2 Isolates Rapidly Evolve In Vitro to Higher Infectivity through More Efficient Binding to Heparan Sulfate and Reduced S1/S2 Cleavage.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.

Natural isolate and recombinant SARS-CoV-2 rapidly evolve in vitro to higher infectivity through more efficient binding to heparan sulfate and reduced S1/S2 cleavage.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo . IMPORTANCE: The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to ACE2 receptor and later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the N-terminal domain (NTD) and in P1â€Ëœ position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells and decrease GE:PFU ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high affinity interaction of the spikes with the ACE2 receptor.

Water-soluble tocopherol derivatives inhibit SARS-CoV-2 RNA-dependent RNA polymerase.

The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs 1-4 , the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits 5 . Here we show that water-soluble derivatives of α-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-α-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and ß-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of α-tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV-2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp 6 . In summary, we conclude that solubilizing modifications to α-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection 7-9 .

NAP1L1 and NAP1L4 Binding to Hypervariable Domain of Chikungunya Virus nsP3 Protein Is Bivalent and Requires Phosphorylation.

Chikungunya virus (CHIKV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Within the last 2 decades, CHIKV has expanded its presence to both hemispheres and is currently circulating in both Old and New Worlds. Despite the severity and persistence of the arthritis it causes in humans, no approved vaccines or therapeutic means have been developed for CHIKV infection. Replication of alphaviruses, including CHIKV, is determined not only by their nonstructural proteins but also by a wide range of host factors, which are indispensable components of viral replication complexes (vRCs). Alphavirus nsP3s contain hypervariable domains (HVDs), which encode multiple motifs that drive recruitment of cell- and virus-specific host proteins into vRCs. Our previous data suggested that NAP1 family members are a group of host factors that may interact with CHIKV nsP3 HVD. In this study, we performed a detailed investigation of the NAP1 function in CHIKV replication in vertebrate cells. Our data demonstrate that (i) the NAP1-HVD interactions have strong stimulatory effects on CHIKV replication, (ii) both NAP1L1 and NAP1L4 interact with the CHIKV HVD, (iii) NAP1 family members interact with two motifs, which are located upstream and downstream of the G3BP-binding motifs of CHIKV HVD, (iv) NAP1 proteins interact only with a phosphorylated form of CHIKV HVD, and HVD phosphorylation is mediated by CK2 kinase, and (v) NAP1 and other families of host factors redundantly promote CHIKV replication and their bindings have additive stimulatory effects on viral replication. IMPORTANCE Cellular proteins play critical roles in the assembly of alphavirus replication complexes (vRCs). Their recruitment is determined by the viral nonstructural protein 3 (nsP3). This protein contains a long, disordered hypervariable domain (HVD), which encodes virus-specific combinations of short linear motifs interacting with host factors during vRC assembly. Our study defined the binding mechanism of NAP1 family members to CHIKV HVD and demonstrated a stimulatory effect of this interaction on viral replication. We show that interaction with NAP1L1 is mediated by two HVD motifs and requires phosphorylation of HVD by CK2 kinase. Based on the accumulated data, we present a map of the binding motifs of the critical host factors currently known to interact with CHIKV HVD. It can be used to manipulate cell specificity of viral replication and pathogenesis, and to develop a new generation of vaccine candidates.

Consequences of Phosphorylation in a Mononegavirales Polymerase-Cofactor System.

Vesicular stomatitis virus (VSV) is a member of the order Mononegavirales, which consists of viruses with a genome of nonsegmented negative-sense (NNS) RNA. Many insights into the molecular biology of NNS viruses were first made in VSV, which is often studied as a prototype for members of this order. Like other NNS viruses, the VSV RNA polymerase consists of a complex of the large protein (L) and phosphoprotein (P). Recent discoveries have produced a model in which the N-terminal disordered segment of P (PNTD) coordinates the C-terminal accessory domains to produce a "compacted" L conformation. Despite this advancement, the role of the three phosphorylation sites in PNTD has remained unknown. Using nuclear magnetic resonance spectroscopy to analyze the interactions between PNTD and the L protein C-terminal domain (LCTD), we demonstrated our ability to sensitively test for changes in the interface between the two proteins. This method showed that the binding site for PNTD on LCTD is longer than was previously appreciated. We demonstrated that phosphorylation of PNTD modulates its interaction with LCTD and used a minigenome reporter system to validate the functional significance of the PNTD-LCTD interaction. Using an electron microscopy approach, we showed that L bound to phosphorylated PNTD displays increased conformational heterogeneity in solution. Taken as a whole, our studies suggest a model in which phosphorylation of PNTD modulates its cofactor and conformational regulatory activities with L.IMPORTANCE Polymerase-cofactor interactions like those addressed in this study are absolute requirements for mononegavirus RNA synthesis. Despite cofactor phosphorylation being present in most of these interactions, what effect if any it has on this protein-protein interaction had not been addressed. Our study is the first to address the effects of phosphorylation on P during its interactions with L in residue-by-residue detail. As phosphorylation is the biologically relevant state of the cofactor, our demonstration of its effects on L conformation suggest that the structural picture of L during infection might be more complex than previously appreciated.

Structural characterization of HIV-1 matrix mutants implicated in envelope incorporation.

During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of MA (A45E, T70R, and L75G) were found to impair Env incorporation and infectivity, leading to the hypothesis that MA trimerization is an obligatory step for Env incorporation. Conversely, Env incorporation can be rescued by a compensatory mutation in the MA trimer interface (Q63R). The impact of these MA mutations on the structure and trimerization properties of MA is not known. In this study, we employed NMR spectroscopy, X-ray crystallography, and sedimentation techniques to characterize the structure and trimerization properties of HIV-1 MA A45E, Q63R, T70R, and L75G mutant proteins. NMR data revealed that these point mutations did not alter the overall structure and folding of MA but caused minor structural perturbations in the trimer interface. Analytical ultracentrifugation data indicated that mutations had a minimal effect on the MA monomer-trimer equilibrium. The high-resolution X-ray structure of the unmyristoylated MA Q63R protein revealed hydrogen bonding between the side chains of adjacent Arg-63 and Ser-67 on neighboring MA molecules, providing the first structural evidence for an additional intermolecular interaction in the trimer interface. These findings advance our knowledge of the interplay of MA trimerization and Env incorporation into HIV-1 particles.