Recombinant Latino virus Pre-glycoprotein polyprotein GP complex (GPC)

What is the basic composition of arenavirus glycoprotein complex?

The arenavirus glycoprotein complex consists of three distinct subunits that function together: the stable signal peptide (SSP), GP1, and GP2. Unlike conventional signal peptides that are degraded after cleavage, the SSP is retained as an essential structural component of the mature complex. The GP1 subunit is primarily responsible for receptor binding, while GP2 mediates membrane fusion. These three subunits interact noncovalently at the virion surface to enable viral attachment and entry into host cells .

How does the arenavirus GPC undergo maturation?

Arenavirus GPC maturation involves two sequential proteolytic cleavage events. First, the stable signal peptide (SSP) is cleaved from the polyprotein precursor by cellular signal peptidase within the endoplasmic reticulum (ER). This 58-amino-acid SSP is retained as a stable subunit. Subsequently, the remaining precursor (GP1/2) is cleaved by the cellular enzyme SKI-1/S1P within the Golgi apparatus, yielding the mature GP1 and GP2 subunits. All three components (SSP, GP1, and GP2) then traffic together to the plasma membrane where they assemble into the functional glycoprotein complex for incorporation into virions .

What distinguishes the stable signal peptide (SSP) of arenaviruses from conventional signal peptides?

Unlike typical signal peptides that are degraded after cleavage, the arenavirus SSP is retained as an essential functional subunit of the mature glycoprotein complex. The SSP plays critical roles beyond ER targeting, including: (1) facilitating proper glycoprotein precursor processing by SKI-1/S1P, (2) enabling trafficking through the secretory pathway, (3) maintaining the native conformation of the glycoprotein complex, and (4) participating in pH-dependent membrane fusion during viral entry. This 58-amino-acid peptide contains a conserved FLLL motif near its C-terminus that is essential for these functions .

What specific role does the conserved FLLL motif in SSP play in glycoprotein complex function?

The highly conserved FLLL motif near the C-terminus of arenavirus SSP serves as a critical sorting signal for glycoprotein maturation and trafficking. Experimental evidence using site-directed mutagenesis demonstrates that mutations in this motif impair multiple aspects of glycoprotein function. The AALL mutation creates a dominant-negative phenotype that prevents glycoprotein expression even when wild-type SSP is provided in trans. The ALLA mutation allows posttranslational modifications but blocks GP2 processing. The FALA mutation permits limited GP2 cleavage that can be partially rescued by wild-type SSP. These findings indicate the FLLL motif is essential for proper glycoprotein folding, ER exit, Golgi processing, and ultimately membrane fusion capacity .

How does the interaction between SSP and GP2 contribute to pH-dependent membrane fusion?

The interaction between SSP and the GP2 subunit is critical for the pH-dependent conformational changes required for membrane fusion. Immunoprecipitation studies confirm direct physical association between SSP and GP2. Mutations in the conserved FLLL motif of SSP disrupt this interaction and significantly impair fusion activity. The SSP-GP2 interaction appears to stabilize the pre-fusion conformation of the glycoprotein complex at neutral pH while allowing the necessary conformational changes triggered by acidification in the endosome. This molecular interplay enables precise control of the fusion process during viral entry, ensuring fusion occurs only in the appropriate cellular compartment .

What is the mechanism of receptor switching during arenavirus entry?

Arenaviruses exhibit a sophisticated receptor switching mechanism during entry. Initially, the GP1 subunit mediates attachment to the primary receptor α-dystroglycan (α-DG) at the cell surface. This interaction triggers virion internalization, likely through macropinocytosis. Following endocytosis and acidification, the virus undergoes a pH-dependent switch from binding α-DG to engaging the lysosomal receptor LAMP1. This secondary receptor binding event is crucial, as it triggers the dissociation of GP1 from the complex, exposing the fusion peptide of GP2 and enabling membrane fusion to occur. This sequential receptor usage ensures fusion happens only in the appropriate endosomal compartment .

What are the optimal expression systems for producing recombinant arenavirus GPCs?

For recombinant expression of arenavirus GPCs, mammalian cell systems typically yield the most native-like glycoproteins. HEK293T cells are frequently used due to their high transfection efficiency and robust protein expression. When designing expression constructs, it is critical to maintain the intact SSP sequence upstream of the GP1/GP2 coding region, as the native SSP is essential for proper folding and processing. For higher yield applications, stable cell lines or lentiviral transduction systems may be preferable to transient transfection. Insect cell expression systems (Sf9, High Five) can produce larger quantities but may result in different glycosylation patterns that could affect function. Codon optimization for the expression host and inclusion of a C-terminal tag that doesn't interfere with SSP-GP2 interactions can improve expression while enabling purification .

How can one assess proper processing and trafficking of recombinant GPC?

Multiple complementary approaches should be used to evaluate recombinant GPC processing and trafficking:

• Western blot analysis using subunit-specific antibodies to detect:

  • Full-length GPC precursor (~75 kDa)

  • Cleaved GP2 subunit (~35 kDa)

  • SSP (~6-10 kDa)

• Flow cytometry of non-permeabilized cells using conformation-specific antibodies against GP1 to quantify surface expression levels.

• Confocal microscopy with organelle markers to track intracellular localization:

  • Calnexin or PDI for ER localization

  • GM130 for Golgi localization

  • Surface staining to confirm plasma membrane trafficking

• Pulse-chase experiments to monitor processing kinetics and trafficking rates.

These methods can reveal defects in specific stages of maturation when comparing wild-type and mutant constructs .

What methodologies are effective for studying SSP-GP2 interactions?

To investigate the critical interactions between SSP and GP2 subunits, researchers can employ several approaches:

• Immunoprecipitation (IP) using epitope-tagged SSP (e.g., HA-tagged) followed by Western blotting for GP2, which can confirm physical association between these subunits.

• Crosslinking studies using membrane-permeable crosslinkers followed by mass spectrometry to identify specific interaction sites.

• Split reporter assays (BiFC, BRET) to visualize interactions in living cells.

• Mutagenesis of key residues in both SSP and the GP2 cytoplasmic domain, followed by functional assays to map interaction interfaces.

• In vitro binding assays using purified peptides corresponding to SSP and the GP2 cytoplasmic domain.

These approaches can identify critical residues and provide insight into how mutations disrupt complex formation and function .

How can membrane fusion activity of recombinant GPC be quantitatively measured?

Several complementary assays can be employed to measure GPC-mediated membrane fusion:

• Syncytium formation assay: Cells expressing GPC are briefly exposed to low pH buffer, then monitored for multinucleated syncytia formation by microscopy. Quantification involves counting nuclei per syncytium or measuring total syncytial area.

• Cell-cell fusion reporter assay: One cell population expressing GPC is co-cultured with target cells containing a reporter activated upon cytoplasmic mixing (e.g., dual-split luciferase or T7 polymerase/T7 promoter systems). Fusion is triggered by low pH and quantified by reporter activity.

• Pseudovirus infectivity assay: Recombinant viral particles bearing the GPC of interest and containing a reporter gene are produced. Entry efficiency correlates with fusion activity and is measured by reporter expression in target cells.

• Direct fusion assays using fluorescent lipid mixing (e.g., R18 dequenching) or content mixing between labeled liposomes and GPC-expressing cells.

These methods allow precise quantification of how mutations affect fusion capacity .

What phenotypic changes occur when the FLLL motif in SSP is mutated?

Mutations in the conserved FLLL motif of SSP produce distinct phenotypes depending on the specific residues altered:

The phenylalanine at position 49 appears particularly critical, as its mutation severely impairs all aspects of glycoprotein function. These phenotypes indicate the FLLL motif is essential for multiple stages of glycoprotein maturation, from initial folding to membrane fusion capability .

How do different arenavirus GPCs vary in their pH threshold for fusion activation?

Arenavirus GPCs exhibit species-specific pH thresholds for fusion activation, which likely reflect adaptations to their particular cellular entry pathways:

• Old World arenaviruses (like Lassa virus and LCMV):

  • Generally activate fusion at pH 5.0-5.5

  • Fusion strictly dependent on receptor switching from α-DG to LAMP1

  • SSP-GP2 interface plays critical role in pH sensing

• New World arenaviruses (including Latino virus):

  • Often trigger fusion at slightly higher pH (5.5-6.0)

  • Some can use transferrin receptor 1 (TfR1) as primary receptor

  • May have distinct SSP-GP2 interaction characteristics

The specific amino acid composition of both the SSP and the GP2 subunit, particularly at their interaction interface, determines this pH threshold. Residues within the SSP transmembrane regions and the conserved FLLL motif are particularly important for setting this threshold .

What strategies can be used to generate chimeric arenavirus GPCs for studying subunit compatibility?

Creating chimeric arenavirus GPCs provides valuable insights into subunit compatibility and functional determinants. Successful chimeric GPC engineering requires careful consideration of the following:

• SSP-GP2 compatibility: The SSP and GP2 cytoplasmic domain must be from the same virus species to maintain proper interactions. Chimeras with mismatched SSP and GP2 cytoplasmic domains typically fail to produce functional glycoproteins.

• Domain swapping approach: Replace discrete domains (SSP, GP1, GP2 ectodomain, or GP2 transmembrane/cytoplasmic domain) while maintaining natural cleavage sites. Use overlap extension PCR to generate seamless junctions.

• Coexpression strategy: Express individual subunits (SSP, GP1/GP2) from separate plasmids and assess complementation.

• Epitope tagging considerations: Add small epitope tags (e.g., HA tag) to track specific subunits, but place these carefully to avoid disrupting critical interactions.

Research has shown that homologous SSP and GP2 cytoplasmic domains are required for infectious virus production, highlighting the specific nature of these interactions .

How can reverse genetics approaches be used to study GPC function in the context of infectious virus?

Reverse genetics systems provide powerful tools for studying GPC function in the authentic context of the viral lifecycle:

• Plasmid-based systems: Most arenavirus reverse genetics systems utilize T7 RNA polymerase-driven expression of viral genomic segments. The S segment containing the GPC can be modified to introduce specific mutations.

• Key considerations for GPC mutations:

  • Mutations that block glycoprotein maturation or trafficking will prevent infectious virus production

  • For lethal mutations, pseudotyped virus systems or trans-complementation approaches may be necessary

  • Temperature-sensitive mutations may allow conditional expression studies

• Rescue strategies for defective GPC:

  • Provide wild-type GPC in trans during initial virus rescue

  • Generate heterozygous virions containing both mutant and wild-type GPC

  • Create replication-competent but propagation-deficient viruses for single-cycle infection studies

• Readouts to assess GPC function:

  • Virus production titers

  • Plaque morphology

  • Growth kinetics

  • Cell-to-cell spread efficiency

These approaches have revealed that residues at positions 5 and 50 within the SSP (the latter residing within the FLLL motif) play crucial roles in viral pathogenicity .

What are the current challenges in generating stable recombinant GPC trimers for structural studies?

Obtaining stable, properly folded arenavirus GPC trimers for structural studies presents several challenges:

• Intrinsic instability of the pre-fusion conformation:

  • The metastable nature of the pre-fusion state makes it prone to triggering

  • GP1 dissociates readily from GP2 under conditions used for purification

• Technical challenges:

  • Maintaining the essential SSP-GP1-GP2 interaction during purification

  • Preserving native trimeric assembly without aggregation

  • Removing detergents without triggering conformational changes

• Potential solutions:

  • Introduction of stabilizing mutations at the GP1-GP2 interface

  • Use of GP1-GP2 crosslinking strategies (disulfide engineering)

  • Addition of conformation-specific antibody Fabs during purification

  • GlycoDelete engineering to reduce glycan heterogeneity while maintaining essential glycans

  • Nanodiscs or amphipol systems to maintain membrane environment

• Expression system considerations:

  • Mammalian expression systems (particularly HEK293 GnTI- cells) provide most native-like glycosylation

  • Insect cell systems may provide higher yield but with altered glycosylation

  • SSP must be co-expressed for proper folding and assembly

Recent structural studies have successfully employed combinations of these approaches to capture the native trimeric pre-fusion conformation of arenavirus GPCs .

How can knowledge of SSP-GP2 interactions inform development of fusion inhibitors?

Understanding the critical SSP-GP2 interaction provides several avenues for developing targeted fusion inhibitors:

• Targeting the SSP-GP2 interface:

  • Small molecules that disrupt the interaction between SSP and GP2 could prevent the conformational changes required for fusion

  • The conserved FLLL motif represents a potential binding site for inhibitors

  • Peptides derived from either SSP or the GP2 cytoplasmic domain could competitively inhibit this interaction

• Stabilizing the pre-fusion conformation:

  • Compounds that bind across subunit interfaces could lock the complex in its pre-fusion state

  • The pH-sensing mechanism involving SSP-GP2 interaction could be targeted to prevent acid-induced triggering

• Screening strategies:

  • Cell-based fusion assays can identify compounds that block pH-dependent fusion

  • Split-reporter systems monitoring SSP-GP2 interaction could identify disruptors

  • In silico docking to structural models of the SSP-GP2 interface

These approaches offer potential for broadly active inhibitors, as the SSP-GP2 interaction mechanism is conserved across arenaviruses .

What considerations are important when designing recombinant GPC constructs for vaccine development?

Designing optimal recombinant GPC constructs for arenavirus vaccines requires careful consideration of several factors:

• Inclusion of all three subunits:

  • The complete SSP-GP1-GP2 complex is necessary to present native epitopes

  • Constructs lacking SSP fail to traffic properly and present non-native conformations

• Stability enhancements:

  • Introduction of stabilizing mutations that maintain the pre-fusion conformation

  • Potential for disulfide bonding between GP1 and GP2 to prevent dissociation

  • Trimerization domains to ensure proper oligomeric state

• Antigen presentation format:

  • Soluble ectodomains versus membrane-anchored forms

  • Virus-like particles incorporating properly processed GPC

  • DNA vaccines encoding full-length GPC with intact SSP

• Safety considerations:

  • Mutations in the fusion peptide to reduce potential reactogenicity

  • Modification of immunosuppressive epitopes where identified

  • Glycan engineering to focus immune responses on protective epitopes

Optimizing these parameters is essential for eliciting antibodies that recognize the native viral glycoprotein and provide protection against infection .

How might cryo-electron tomography advance our understanding of native GPC arrangement on virions?

Cryo-electron tomography (cryo-ET) offers unique opportunities to study the native arrangement of GPC trimers in the context of intact virions:

• Technical advantages for arenavirus GPC research:

  • Visualizes glycoprotein complexes in their native membrane environment

  • Reveals the spatial distribution and orientation of GPC trimers on the virion surface

  • Can identify interactions between the GPC and underlying matrix proteins

  • Maintains the critical SSP component often lost in purified preparations

• Research questions addressable through cryo-ET:

  • How densely packed are GPC trimers on the virion surface?

  • Do GPCs cluster or maintain uniform distribution?

  • What is the relationship between GPC trimers and the underlying matrix lattice?

  • Are there conformational differences between GPCs at different locations on the virion?

• Methodological considerations:

  • Sub-tomogram averaging to improve resolution of individual GPC structures

  • Correlative approaches combining fluorescence and electron microscopy

  • Labeling strategies to identify specific domains within the native complex

Previous cryo-ET studies have shown that glycoprotein spike trimers connect to the underlying matrix, suggesting an organized arrangement that may influence fusion dynamics .

What role might artificial intelligence play in predicting the effects of GPC mutations?

Artificial intelligence approaches hold significant promise for predicting how mutations affect arenavirus GPC structure and function:

• Deep learning applications:

  • Predicting the impact of point mutations on GPC processing efficiency

  • Modeling conformational changes during the fusion process

  • Identifying potential interaction sites between subunits

• Machine learning for epitope prediction:

  • Identifying immunodominant versus neutralizing epitopes

  • Predicting cross-reactive epitopes across arenavirus species

  • Optimizing immunogen design by predicting immune responses

• AI-assisted protein engineering:

  • Designing stabilized pre-fusion conformations

  • Optimizing expression and folding of recombinant constructs

  • Predicting the impact of glycosylation site modifications

• Data integration approaches:

  • Combining structural, functional, and evolutionary data to identify critical residues

  • Predicting phenotypic outcomes from genotypic changes observed in field isolates

As more structural and functional data become available for arenavirus GPCs, these AI approaches will become increasingly powerful for guiding experimental design and interpretation .

How does the GPC interact with host factors beyond the primary receptors?

Research on arenavirus GPC interactions with host factors beyond primary receptors reveals complex host-pathogen interactions:

• Cellular factors influencing GPC maturation:

  • ER chaperones (calnexin, calreticulin) that facilitate folding

  • ERGIC-53 involvement in glycoprotein trafficking

  • Components of the COPII vesicle system for ER export

• Host restriction factors:

  • IFITM proteins that can inhibit membrane fusion

  • Tetherin interactions with GPC and potential antagonism

  • Glycan-binding lectins that may modulate GPC function

• Immunological interactions:

  • Recognition by pattern recognition receptors

  • Evasion of complement-mediated neutralization

  • Modulation of NK cell activation through MHC class I downregulation

• Methodologies to identify novel interactors:

  • Proximity labeling approaches (BioID, APEX)

  • Systematic CRISPR screens for host dependency factors

  • Immunoprecipitation coupled with mass spectrometry