Envelope Glycoproteins Gn Research Articles

Interaction between the SFTSV envelope glycoprotein Gn and STING inhibits the formation of the STING-TBK1 complex and suppresses the NF-κB signaling pathway.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly discovered virus associated with severe hemorrhagic fever in humans. However, the role of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway during SFTSV infection is still unclear. We found that SFTSV infection inhibits downstream innate immune signaling pathways by activating the cytoplasmic DNA receptor cGAS. In addition, SFTSV Gn blocked the nuclear accumulation of interferon regulatory factor 3 and p65 to inhibit downstream innate immune signaling. Moreover, we determined that Gn of SFTSV inhibited K27-ubiquitin modification of STING to disrupt the assembly of the STING-TANK-binding kinase 1 complex and downstream signaling. We found that the SFTSV envelope glycoprotein Gn is a potent inhibitor of the cGAS-STING pathway. In conclusion, this study highlights the crucial function of the glycoprotein Gn in the antiviral innate immune response and reveals a new method of immune escape of SFTSV.

The Hantavirus Surface Glycoprotein Lattice and Its Fusion Control Mechanism

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-Å-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gcmembrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses.

High-Resolution Description of the Hantavirus Surface Glycoprotein Lattice and Its Membrane Fusion Control Mechanism

Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. The hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimeric protomers associated into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the Gn head / Gc heterodimer, and of the homotetrameric Gn base. Docking them into an 11.4 A resolution cryo- electron tomography map of the hantavirus surface accounted for the complete extracellular portion of the viral glycoprotein shell and provided unprecedented detail of the surface organization of a pleomorphic virus. These results, which further revealed an in-built mechanism controlling Gc membrane-insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses

Molecular organization and dynamics of the fusion protein Gc at the hantavirus surface.

The hantavirus envelope glycoproteins Gn and Gc mediate virion assembly and cell entry, with Gc driving fusion of viral and endosomal membranes. Although the X-ray structures and overall arrangement of Gn and Gc on the hantavirus spikes are known, their detailed interactions are not. Here we show that the lateral contacts between spikes are mediated by the same 2-fold contacts observed in Gc crystals at neutral pH, allowing the engineering of disulfide bonds to cross-link spikes. Disrupting the observed dimer interface affects particle assembly and overall spike stability. We further show that the spikes display a temperature-dependent dynamic behavior at neutral pH, alternating between 'open' and 'closed' forms. We show that the open form exposes the Gc fusion loops but is off-pathway for productive Gc-induced membrane fusion and cell entry. These data also provide crucial new insights for the design of optimized Gn/Gc immunogens to elicit protective immune responses.

Structural Transitions of the Conserved and Metastable Hantaviral Glycoprotein Envelope.

ABSTRACTHantaviruses are zoonotic pathogens that cause severe hemorrhagic fever and pulmonary syndrome. The outer membrane of the hantavirus envelope displays a lattice of two glycoproteins, Gn and Gc, which orchestrate host cell recognition and entry. Here, we describe the crystal structure of the Gn glycoprotein ectodomain from the Asiatic Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Structural overlay analysis reveals that the HTNV Gn fold is highly similar to the Gn of Puumala virus (PUUV), a genetically and geographically distinct and less pathogenic hantavirus found predominantly in northeastern Europe, confirming that the hantaviral Gn fold is architecturally conserved across hantavirus clades. Interestingly, HTNV Gn crystallized at acidic pH, in a compact tetrameric configuration distinct from the organization at neutral pH. Analysis of the Gn, both in solution and in the context of the virion, confirms the pH-sensitive oligomeric nature of the glycoprotein, indicating that the hantaviral Gn undergoes structural transitions during host cell entry. These data allow us to present a structural model for how acidification during endocytic uptake of the virus triggers the dissociation of the metastable Gn-Gc lattice to enable insertion of the Gc-resident hydrophobic fusion loops into the host cell membrane. Together, these data reveal the dynamic plasticity of the structurally conserved hantaviral surface.IMPORTANCE Although outbreaks of Korean hemorrhagic fever were first recognized during the Korean War (1950 to 1953), it was not until 1978 that they were found to be caused by Hantaan virus (HTNV), the most prevalent pathogenic hantavirus. Here, we describe the crystal structure of HTNV envelope glycoprotein Gn, an integral component of the Gn-Gc glycoprotein spike complex responsible for host cell entry. HTNV Gn is structurally conserved with the Gn of a genetically and geographically distal hantavirus, Puumala virus, indicating that the observed α/β fold is well preserved across the Hantaviridae family. The combination of our crystal structure with solution state analysis of recombinant protein and electron cryo-microscopy of acidified hantavirus allows us to propose a model for endosome-induced reorganization of the hantaviral glycoprotein lattice. This provides a molecular-level rationale for the exposure of the hydrophobic fusion loops on the Gc, a process required for fusion of viral and cellular membranes.

N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)–any amino acid (X)–serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: “Gc-large” and “Gc-small”, and N1077 was responsible for “Gc-large” band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN.

Analysis of the humoral immune response against the envelope glycoprotein Gc of Schmallenberg virus reveals a domain located at the amino terminus targeted by mAbs with neutralizing activity

Orthobunyaviruses are enveloped viruses that are arthropod-transmitted and cause disease in humans and livestock. Viral attachment and entry are mediated by the envelope glycoproteins Gn and Gc, and the major glycoprotein, Gc, of certain orthobunyaviruses is targeted by neutralizing antibodies. The domains in which the epitopes of such antibodies are located on the glycoproteins of the animal orthobunyavirus Schmallenberg virus (SBV) have not been identified. Here, we analysed the reactivity of a set of mAbs and antisera against recombinant SBV glycoproteins. The M-segment-encoded proteins Gn and Gc of SBV were expressed as full-length proteins, and Gc was also produced as two truncated forms, which consisted of its amino-terminal third and carboxyl-terminal two-thirds. The sera from convalescent animals reacted only against the full-length Gc and its subdomains and not against the SBV glycoprotein Gn. Interestingly, the amino-terminal domain of SBV-Gc was targeted not only by polyclonal sera but also by the majority of murine mAbs with a neutralizing activity. Furthermore, the newly defined amino-terminal domain of about 230 aa of the SBV Gc protein could be affinity-purified and further characterized. This major neutralizing domain might be relevant for the development of prophylactic, diagnostic and therapeutic approaches for SBV and other orthobunyaviruses.

The cytoplasmic tail of hantavirus Gn glycoprotein interacts with RNA

We recently characterized the interaction between the intraviral domains of envelope glycoproteins (Gn and Gc) and ribonucleoprotein (RNP) of Puumala and Tula hantaviruses (genus Hantavirus, family Bunyaviridae). Herein we report a direct interaction between spike-forming glycoprotein and nucleic acid. We show that the envelope glycoprotein Gn of hantaviruses binds genomic RNA through its cytoplasmic tail (CT). The nucleic acid binding of Gn-CT is unspecific, as demonstrated by interactions with unrelated RNA and with single-stranded DNA. Peptide scan and protein deletions of Gn-CT mapped the nucleic acid binding to regions that overlap with the previously characterized N protein binding sites and demonstrated the carboxyl-terminal part of Gn-CT to be the most potent nucleic acid-binding site. We conclude that recognition of the RNP complex by the Gn-CT could be mediated by interactions with both genomic RNA and the N protein. This would provide the required selectivity for the genome packaging of hantaviruses.

Cytomegalovirus gN Genotypes Distribution among Congenitally Infected Newborns and Their Relationship with Symptoms at Birth and Sequelae

Cytomegalovirus (CMV) is the leading cause of congenital infection, with morbidity and mortality at birth and sequelae. Both host and viral factors may affect the outcome of infection. CMV strain virulence may depend on genetic variability in "key genes," such as UL73, which encodes the envelope glycoprotein gN. This study aimed to ascertain the role of gN variants as markers of pathogenicity and prognosis in newborns congenitally infected with CMV. Seventy-four congenitally infected newborns were monitored for symptoms of CMV disease at birth and during long-term follow-up. The distribution of gN variants was analyzed in relation to virological parameters, clinical signs, laboratory and instrumental abnormalities at birth, and sequelae. Multivariate cluster analysis was used to test for differences in the distribution of variables. An independent validation cohort of the same size and modality of recruitment as the original population was examined by logistic regression to validate results. Univariate and cluster analyses suggest that newborns congenitally infected with CMV fall into 2 subpopulations on the basis of definite parameters of CMV disease. The first population with no symptoms at birth, negative instrumental findings, and a favorable long-term outcome was significantly associated with gN-1 and gN-3a genotypes. The second group with symptoms at birth, abnormal imaging results, and sequelae was associated with gN-4 genotypes (P<.05). The validation cohort further supports the results, indicating that genotypes gN-1 or gN-3a reduce the risk of sequelae 5 fold (95% confidence interval, 1.3-15.6 fold), whereas variants belonging to group gN-4 increase the risk of sequelae 8 fold (95% confidence interval, 2.6-25.8 fold). Results suggest that gN genotypes might be markers for virulence of CMV wild-type strains and a discriminating factor for selection of CMV-infected newborns who are at risk of developing sequelae.

Degradation and aggresome formation of the Gn tail of the apathogenic Tula hantavirus

The cytoplasmic tails of envelope glycoprotein Gn of pathogenic hantaviruses but not of the apathogenic Prospect Hill virus (PHV) were recently reported to be proteasomally degraded in simian COS7 cells. Here, we show that the cytoplasmic tails of the glycoproteins of the apathogenic hantaviruses Tula virus (TULV) and PHV are also degraded through the ubiquitin-proteasome pathway, both in human HEK-293 and in simian Vero E6 cells. TULV Gn tails formed aggresomes in cells with proteasomal inhibitors. We conclude that degradation upon aggregation of Gn tails, which may represent a general cellular response to misfolded protein used by hantaviruses to control maturation of virions, is unrelated to pathogenicity.

Requirements for ER‐Arrest and Sequential Exit to the Golgi of Tomato Spotted Wilt Virus Glycoproteins

The envelope glycoproteins Gn and Gc are major determinants in the assembly of Tomato spotted wilt virus (TSWV) particles at the Golgi complex. In this article, the ER-arrest of singly expressed Gc and the transport of both glycoproteins to the Golgi upon coexpression have been analyzed.While preliminary results suggest that the arrest of Gc at the ER (endoplasmic reticulum) did not appear to result from improper folding, transient expression of chimeric Gc, in which the transmembrane domain (TMD) and/or cytoplasmic tail (CT) were swapped for those from Gn, showed that the TMD of Gn was sufficient to allow ER exit and transport to the Golgi. Expression of both glycoproteins in the presence of overexpressed Sar1p specific guanosine nucleotide exchange factor Sec12p, resulted in ER-retention demonstrating that the viral glycoproteins are transported to the Golgi in a COPII (coat protein II)-dependent manner. Inhibition of ER Golgi transport by brefeldin A (BFA) had a similar effect on the localization of Gn. However, inhibition of ER (endoplasmic reticulum) to Golgi transport of coexpressed Gc and Gn by overexpression of Sec12p or by BFA revealed distinct localization patterns, i.e. diffuse ER localization versus concentration at specific spots.

A Soluble Form of the Tomato spotted wilt virus (TSWV) Glycoprotein GN (GN-S) Inhibits Transmission of TSWV by Frankliniella occidentalis

Tomato spotted wilt virus (TSWV) is an economically important virus that is transmitted in a persistent propagative manner by its thrips vector, Frankliniella occidentalis. Previously, we found that a soluble form of the envelope glycoprotein G(N) (G(N)-S) specifically bound thrips midguts and reduced the amount of detectable virus inside midgut tissues. The aim of this research was to (i) determine if G(N)-S alters TSWV transmission by thrips and, if so, (ii) determine the duration of this effect. In one study, insects were given an acquisition access period (AAP) with G(N)-S mixed with purified virus and individual insects were assayed for transmission. We found that G(N)-S reduced the percent of transmitting adults by eightfold. In a second study, thrips were given an AAP on G(N)-S protein and then placed on TSWV-infected plant material. Individual insects were assayed for transmission over three time intervals of 2 to 3, 4 to 5, and 6 to 7 days post-adult eclosion. We observed a significant reduction in virus transmission that persisted to the same degree throughout the time course. Real-time reverse transcription polymerase chain reaction analysis of virus titer in individual insects revealed that the proportion of thrips infected with virus was reduced threefold when insects were preexposed to the G(N)-S protein as compared to no exposure to protein, and nontransmitters were not infected with virus. These results demonstrate that thrips transmission of a tospovirus can be reduced by exogenous viral glycoprotein.

Comparative analysis of the alphavirus-based vectors expressing Rift Valley fever virus glycoproteins

During the last decade, alphaviruses became widely used for expression of heterologous genetic information and development of recombinant vaccines against a variety of human and animal pathogens. In this study, we compared a number of vectors based on the genome of Sindbis (SINV) and Venezuelan equine encephalitis (VEEV) viruses for their ability to express the Rift Valley fever virus (RVFV) envelope glycoprotein Gn and induce a protective immune response against RVFV infection. Our results suggest that (i) application of VEEV-based expression systems appears to be advantageous, when compared to similar systems designed on the basis of the SINV genome. (ii) Alphavirus-specific E3 and E2 proteins and furin-specific cleavage sites can be used for engineering secreted forms of the proteins. (iii) Alphaviruses can be modified for expression of the large fragments of heterologous proteins on the surface of chimeric, infectious viral particles. Thus, alphavirus-based expression systems may have the potential for a broader application beyond their current use as replicons or double-subgenomic vectors.

Genomic variants among human cytomegalovirus (HCMV) clinical isolates: the glycoprotein n (gN) paradigm

Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms, which are supposed to be implicated in strain-specific tissue tropism and HCMV-induced immunopathogenesis. One highly variable gene is ORF UL73, encoding for the envelope glycoprotein gN, which displays both a structural and an immunologic role as a component of the high-molecular weight complex gC-II. UL73 showed clustered polymorphisms, which originate four distinct genomic variants, denoted gN-1, gN-2, gN-3, and gN-4. This review reports the main features of gN genotypes and their potential implications on HCMV biologic properties. The clinical impact of gN variants is also discussed. This overview on gN clustered polymorphisms should be useful as a prototype model for a better understanding of the biologic and clinical relevance of HCMV clinical isolates genetic variability.

Intrauterine cytomegalovirus infection and glycoprotein N (gN) genotypes.

Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms, supposed to be related with strain-specific tissue-tropism and HCMV-induced immunopathogenesis. One recently discovered polymorphic gene is ORF UL73, encoding for the envelope glycoprotein gN. Among HCMV clinical strains, it shows four distinct genomic variants denoted as gN-1, gN-2, gN-3 and gN-4. Aims of this study were to assess the prevalence of the different gN types in the populations examined and to investigate the possible relationship between genotypes and severity of congenital CMV disease. The gN genotyping was carried out by sequencing analysis of the HCMV ORF UL73. Comparisons were made by chi-square test and contingency tables. All the four gN genotypes can cause congenital infections and the overall distribution was as follows: gN-1, 23.6%; gN-2, 1.1%; gN-3, 12.9%; gN-4, 62.4%. None of them seems to be preferentially associated with vertical transmission or with acute outcome of congenital infection. However, considering the chronic outcome and long-term sequelae, there was a statistically significant (P<0.05) difference between congenitally infected infants with or without adverse chronic outcome. HCMV congenital infections, which displayed a prevalence of the gN-1 variants, seem to be associated with favorable chronic outcome.