Recombinant Lymphocytic choriomeningitis virus Pre-glycoprotein polyprotein GP complex (GPC)

What is the structure of the LCMV glycoprotein complex and how does it facilitate viral entry?

The LCMV glycoprotein complex (GPC) consists of several components that work together to facilitate viral entry. The complex includes a membrane-associated signal peptide and two primary subunits: GP1, which is responsible for receptor binding, and GP2, which mediates fusion with the host cell membrane . The crystal structure of the prefusion GP1-GP2 complex has been resolved at 3.5Å, revealing intimate association between these subunits with approximately 5300Ų of surface area buried at their interface .

The GP1-GP2 interface can be mapped to four primary regions:

• The N-terminal portion of GP1 contacts HR2 of GP2

• β1 of GP1 assembles with the T-loop strands β10 and β11 of GP2

• F2 contacts β1 and the loop connecting β1 and β2 of GP1

• α4 of GP1 occupies a cleft between HR1c and HR1d in GP2, anchoring these helices in the pre-fusion state

Electrostatic analysis reveals that the β-sheet surface of GP1 is primarily acidic, while both the lower helix-loop face of GP1 and the GP1-GP2 assembly sites have a largely basic surface . This structural arrangement is critical for the conformational changes the glycoprotein must undergo during the fusion process.

What cellular receptors are involved in LCMV entry and how do they interact with the viral glycoprotein?

LCMV entry involves multiple cellular receptors that interact with the viral glycoprotein in a sequential manner. Initially, LCMV GP interacts with α-dystroglycan (α-DG) and heparan sulfate at the plasma membrane . Recent research has identified that after endocytosis, as the pH drops in endolysosomal compartments, the virus switches to utilizing CD164, a lysosomal sialomucin, as a critical receptor .

Importantly, the full-length GPC, rather than just the GP1 subunit alone, is required for efficient binding to α-DG. This was demonstrated through immunoprecipitation studies with cells overexpressing the LARGE glycosyltransferase necessary for correct modification of the receptor . The requirement for the complete GPC structure suggests a complex interaction mechanism that likely involves conformational elements spanning both GP1 and GP2 subunits.

The central cysteine-rich domain (CRD) of CD164 and a specific sialylated N-linked glycan within that domain are required for LCMV infection . The interaction between purified GP and CD164 is pH-dependent, occurring preferentially at acidic pH levels found in endolysosomal compartments, which facilitates the membrane fusion process necessary for viral entry .

How does the LCMV glycoprotein complex undergo conformational changes during the fusion process?

The prefusion and postfusion forms of LCMV GP2 exhibit striking structural differences that illuminate the fusion mechanism. In the stable prefusion conformation, GP2 maintains a specific arrangement of helical regions and fusion peptides that prevent premature fusion . Following acidification in the endosome, substantial conformational rearrangements occur.

In the pH-induced post-fusion conformation, the heptad repeat regions HR1 and HR2 each form a single helix, with a 41-residue "T loop" positioned between them . Three copies of each gather to form an antiparallel six-helix bundle (6HB), a structural arrangement that is critical for pulling the viral and cellular membranes together to facilitate fusion .

The fusion mechanism involves two distinct fusion elements in GP2:

• The F1 N-terminal peptide, which is partly helical and similar in structure to the fusion peptide of parainfluenza 5 virus F

• The F2 internal loop, which uses an anti-parallel β strand scaffold to display a hydrophobic fusion segment at its center, similar to the fusion loops of Ebola virus GP and those of class II and III viruses

This dual fusion element arrangement suggests a complex fusion mechanism that may involve multiple contact points between the viral and host cell membranes.

What approaches have been used to develop recombinant LCMV vectors, and what are their advantages for research applications?

Researchers have successfully developed recombinant LCMV vectors through several sophisticated approaches. One notable strategy involved the generation of a recombinant S segment RNA (Sr) where the glycoprotein of vesicular stomatitis virus (VSVG) was substituted for the LCMV glycoprotein . This recombinant construct was produced intracellularly from cDNA under the control of a polymerase I promoter, with coexpression of LCMV proteins NP and L enabling expression of VSVG from the Sr .

The isolation of recombinant LCMV expressing VSVG (rLCMV/VSVG) was achieved through a clever selection strategy against wild-type LCMV (LCMVwt) using a cell line deficient in the cellular protease S1P . This approach exploited the finding that processing of LCMV-GP by S1P is required for virus infectivity, whereas VSVG function is independent of this processing .

For experimental applications, rLCMV/VSVG offers several advantages:

• It enables investigation of whether the low cytotoxicity of LCMV-GP plays a key role in maintaining virus-host balance during persistence

• The surface VSVG serves as an excellent target for neutralizing antibodies

• It can function as a helper virus to efficiently generate additional S segment rLCM viruses

Despite expressing a heterologous glycoprotein, rLCMV/VSVG maintains key biological properties of the parent virus, including the ability to persist in neonatally infected mice without clinical signs of disease, making it valuable for studying mechanisms of viral persistence .

How can LCMV GPC be modified to enhance its immunogenicity for vaccine development purposes?

LCMV GPC can be significantly modified to enhance its immunogenicity through structural alterations that affect protein stability, subcellular localization, and processing. One highly successful approach involved creating a C-terminally truncated, noncleavable variant of LCMV-GP that led to the accumulation of stable, soluble GP trimers in the endoplasmic reticulum (ER) of antigen donor cells .

This modified variant, termed GPER, differs from wild-type GP in several key ways:

• Lack of a transmembrane domain

• Addition of a phage-derived trimerization-promoting domain

• Resistance to processing into metastable GP-1/GP-2 complexes due to a mutated protease cleavage site

These modifications resulted in dramatic enhancement of cross-presentation efficiency, with the variant showing >3-4 orders of magnitude improvement in the ability to cross-prime cytotoxic T lymphocyte (CTL) responses compared to wild-type GP . The enhanced immunogenicity cannot be attributed solely to the approximately 10-fold higher protein expression levels but appears to result from the combination of soluble, noncleavable, trimer-stabilized GP complexes and their retention in the ER .

Immunization studies demonstrated that tumor cells expressing the ER-retained LCMV-GP variant cross-primed protective antiviral CTL responses in vivo that conferred full protection against viral challenge . Importantly, these cross-primed CTLs remained functional during both the effector phase (day 15 after immunization) and memory phase (day 41 after immunization) .

What is known about the oligomeric state of LCMV GPC, and how does this affect function and immunogenicity?

The oligomeric state of LCMV GPC plays a crucial role in its function and immunogenicity. Size exclusion chromatography in tandem with multi-angle light scattering (SEC-MALS) has demonstrated that recombinant LCMV GPe forms stable dimers . Crystal structure analysis revealed that the asymmetric unit contains two copies of LCMV GPe in a 2-fold related antiparallel dimer covering a total of 4300 Ų buried surface .

The core of this dimeric interaction consists of a four-helix bundle created by:

• Two copies of α2 in GP1

• Two copies of HR1c in GP2

Notably, the GP2 fusion peptide F1 is buried at the dimer interface, where it bridges across to GP1 of the opposing protomer, forming an extensive hydrogen bond network and additional hydrophobic interactions . This arrangement is reminiscent of the flavivirus envelope protein E, suggesting potential functional similarities in the fusion mechanism .

Research with modified variants has shown that enhancing the trimerization of GP through the addition of a phage-derived trimerization-promoting domain can significantly boost immunogenicity . The stable trimeric structure of the modified GPER variant appears to be more efficiently processed through the cross-presentation pathway, leading to dramatically enhanced CTL priming compared to wild-type GP . This suggests that the natural metastability of GP1/GP2 complexes may contribute to the typically poor cross-presentation of LCMV-GP.

What screening approaches can identify host factors critical for LCMV entry?

A comprehensive genome-wide CRISPR-Cas9 loss-of-function screening approach has proven highly effective for identifying host factors critical for LCMV entry. This methodology employs a vesicular stomatitis virus recombinant in which the native glycoprotein is replaced with that of LCMV . The screen is typically conducted in human cell lines such as the lung epithelial A549 cells, which are permissive to LCMV infection .

The screening workflow involves:

• Generation of a genome-wide CRISPR knockout library in target cells

• Infection of the cell library with the recombinant virus

• Selection for cells resistant to infection

• Sequencing of guide RNAs enriched in the surviving population

• Bioinformatic analysis to identify candidate genes

Following the identification of candidate genes, validation is performed through combined biochemical, genetic, and virological approaches. For the CD164 host factor, validation included:

• Confirming the requirement for the central cysteine-rich domain (CRD)

• Identifying specific N-linked glycans necessary for virus interaction

• Demonstrating direct binding of purified GP to CD164 at acidic pH

• Showing that this interaction results in membrane fusion

This systematic approach has successfully uncovered previously unknown host factors involved in LCMV entry that represent candidate therapeutic targets for combating LCMV infection.

How can researchers effectively produce and purify recombinant LCMV glycoproteins for structural and functional studies?

Production and purification of recombinant LCMV glycoproteins for structural and functional studies require specialized approaches to maintain proper folding, post-translational modifications, and stability. Based on successful structural studies, the following methodological pipeline has proven effective:

• Construct Design:

  • For structural studies, design a recombinant expression construct (e.g., GPe) that includes the ectodomains of GP1 and GP2 while excluding the transmembrane domain

  • Maintain the critical GP1-GP2 interface by ensuring the construct includes both subunits

  • For studying specific functions, more targeted constructs may be designed (e.g., soluble GP1 for receptor binding studies)

• Expression System:

  • Mammalian expression systems are preferred to ensure proper glycosylation and disulfide bond formation

  • Successful expression has been achieved using human embryonic kidney cells (HEK293T)

  • Consider using secretion signals to facilitate protein export into the culture medium

• Purification Strategy:

  • Implement multi-step purification protocols that typically include:
    a. Affinity chromatography (e.g., using His-tags or Fc-fusion approaches)
    b. Size exclusion chromatography to separate properly folded oligomers from aggregates
    c. Ion exchange chromatography for final polishing

  • Validate purified protein by size exclusion chromatography in tandem with multi-angle light scattering (SEC-MALS) to confirm oligomeric state

• Stability Enhancement:

  • For proteins intended for crystallization or long-term studies, consider stability-enhancing modifications such as:
    a. Addition of trimerization domains
    b. Mutation of protease cleavage sites to prevent processing
    c. Strategic disulfide bond engineering

Following these methodological approaches has enabled successful production of LCMV glycoproteins that retain their native conformations and functional properties, facilitating breakthrough structural studies like the 3.5Å crystal structure of the prefusion GP1-GP2 complex .

What assays are available to evaluate LCMV glycoprotein-mediated entry and fusion?

Several sophisticated assays have been developed to evaluate LCMV glycoprotein-mediated entry and fusion, each providing unique insights into different aspects of the entry process:

• Pseudotype Virus Assays:

  • Vesicular stomatitis virus (VSV) recombinants with the native glycoprotein replaced by LCMV-GP provide a versatile system for entry studies

  • These pseudotyped viruses can be engineered to express reporter genes such as chloramphenicol acetyltransferase (CAT) to quantitatively measure entry efficiency

  • Comparison between wild-type and mutant glycoproteins in this system allows for assessment of specific domain functions

• Virus-Like Particle (VLP) Assays:

  • VLPs containing LCMV glycoproteins can be generated by co-expression of viral structural proteins

  • Reporter genes packaged within these VLPs enable quantification of entry events

  • This system has demonstrated that heterologous glycoproteins like VSVG can mediate infectivity of LCMV VLPs with comparable efficiency to LCMV-GP

• Direct Binding Assays:

  • Biochemical approaches to measure direct binding between purified GP and putative receptors

  • Techniques include co-immunoprecipitation assays, surface plasmon resonance, and ELISA-based methods

  • For CD164 studies, binding was demonstrated to occur preferentially at acidic pH, mimicking conditions in endolysosomal compartments

• Cell-Cell Fusion Assays:

  • Syncytium formation can serve as a visual indicator of GP-mediated membrane fusion

  • rLCMV/VSVG has been observed to cause syncytium formation in cultured cells, unlike wild-type LCMV

  • Quantitative fusion assays using split reporter systems can provide more precise measurements of fusion efficiency

• Genetic Screens:

  • Genome-wide CRISPR-Cas9 screens provide a powerful approach to identify host factors required for viral entry

  • This approach successfully identified CD164 as a critical host factor for LCMV entry

  • Similar screening approaches can be adapted to identify factors involved in specific steps of the entry process

These methodological approaches provide researchers with a comprehensive toolkit for dissecting the complex process of LCMV glycoprotein-mediated entry and fusion, enabling the identification of potential therapeutic targets and advancing our understanding of arenavirus biology.