HSV-2 gG
Description
Introduction to HSV-2 gG
Herpes Simplex Virus type 2 (HSV-2) glycoprotein G (gG-2) is a viral envelope protein critical for type-specific serodiagnosis and immune evasion. Unlike other HSV glycoproteins, gG-2 undergoes post-translational cleavage into two distinct portions: a secreted amino-terminal fragment and a cell-associated, heavily O-glycosylated carboxy-terminal mature gG-2 (mgG-2) . This unique processing enables mgG-2 to serve as a primary target for neutralizing antibodies and a key antigen for distinguishing HSV-2 from HSV-1 infections .
Protein Processing
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Precursor Cleavage: The full-length gG-2 precursor is cleaved cotranslationally, producing a secreted portion (detected in viral supernatants) and a cell-associated mgG-2 (detected in infected cells) .
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Glycosylation: The mature form undergoes extensive O-glycosylation, contributing to its immunogenicity .
Genetic Mutations and Phenotypic Variants
Clinical HSV-2 isolates lacking mgG-2 expression are rare but documented. These "gG-2-negative" strains arise from frameshift mutations in the gG-2 gene, typically within GC-rich regions, leading to premature termination codons . For example:
| Mutation Type | Impact on gG-2 Expression | Clinical Isolate Example |
|---|---|---|
| Frameshift (insertion) | No protein expression | Strains 2434, 512, 147 |
| Frameshift (deletion) | Truncated mgG-2 (partial expression) | Strain VI-4444 |
Such mutations disrupt antibody detection, complicating serodiagnosis .
Antibody Targeting and Serodiagnosis
mgG-2 is the primary antigen for type-specific HSV-2 detection due to its absence in HSV-1 and high specificity . Key diagnostic assays include:
| Assay Type | Antigen Used | Sensitivity | Specificity |
|---|---|---|---|
| FOCUS2 ELISA | Recombinant sgG-2 | 97% (GUD) | 74% (Blood) |
| sgG-2 ELISA | Secreted gG-2 | 97% (GUD) | 89% (Blood) |
| mgG-2 ELISA | Native mgG-2 | 99% (GUD) | 97% (Blood) |
GUD: Genital Ulcer Disease cohort; Blood: Blood donor cohort .
Challenges in Serodiagnosis
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False-Negative Results: Patients infected with gG-2-negative HSV-2 strains may lack detectable antibodies, complicating diagnosis .
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Cross-Reactivity: Recombinant sgG-2 (e.g., in FOCUS2) shows lower specificity compared to native mgG-2 .
Preclinical Vaccine Studies
Immunization with mgG-2 + CpG adjuvant in mice demonstrated:
| Parameter | mgG-2 + CpG | mgG-2 Alone | Control |
|---|---|---|---|
| Survival Rate | 73% | 20% | 0% |
| Disease Score | Low | Moderate | Severe |
| Viral Load (Spinal Cord) | Limited | High | High |
Protection correlated with IFN-γ-producing CD4+ T cells .
Mechanisms of Immune Evasion
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Antigenic Variation: gG-2 escape mutants (e.g., strains unreactive to multiple MAbs) evade antibody responses, necessitating multi-epitope targeting in vaccines .
Role in HSV-2 Pathogenesis
While gG-2 is dispensable for in vitro replication, its rarity in clinical isolates suggests in vivo functional importance, potentially in immune modulation or viral entry .
Future Directions
Product Specs
Q&A
What is the molecular structure and processing of HSV-2 glycoprotein G?
HSV-2 glycoprotein G (gG-2) is a viral envelope protein with unique processing characteristics. The gG-2 precursor protein undergoes cotranslational glycosylation, generating a high-mannose intermediate which is then cleaved during processing to produce two distinct portions:
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A secreted amino-terminal portion
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A cell- and virion-associated, heavily O-glycosylated carboxy-terminal portion (mature gG-2)
The first 22 N-terminal amino acids constitute the signal sequence, which is removed during post-translational processing. Amino acid sequencing of the secreted portion identified the N-terminal sequence as GSGVPGPI, corresponding to positions 23-30 after the start codon .
Why is gG-2 significant for HSV serological differentiation?
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The only significant amino acid similarity between gG-1 (HSV-1) and gG-2 (HSV-2) occurs at the signal sequence and in the short membrane anchor
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Since the signal sequence is removed during posttranslational processing and the membrane anchor is sequestered by the lipid bilayer, only the unrelated portions of the gG molecules are available as antigenic epitopes
Is the gG-2 gene essential for HSV-2 replication and pathogenesis?
The gG-2 gene appears to be dispensable for HSV-2 replication, similar to other envelope glycoprotein genes:
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Viable gG-2-deficient mutant viruses have been successfully constructed in vitro
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Clinical HSV-2 isolates with inactivated gG-2 genes have been identified in non-immunocompromised patients with recurrent infections
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These strains can reactivate in vivo to cause clinical lesions, suggesting the gG-2 gene may be classified as nonessential in vivo, at least in some hosts
What techniques can detect expression of gG-2 protein products in HSV-2 isolates?
Researchers can employ several complementary methods to detect gG-2 expression:
Immunoblotting for carboxy-terminal portion:
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Infect HEp-2 cells with HSV-2 isolates
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Harvest cells upon complete cytopathic effect
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Lyse cells in Tris-buffered saline with 1% Nonidet P-40, followed by ultrasonication
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Subject to SDS-PAGE using 10% Tris-glycine gel
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Electrotransfer proteins to membrane
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Incubate with anti-gG-2 monoclonal antibodies (e.g., O1.C5.B2)
In wild-type HSV-2, both the carboxy-terminal high-mannose intermediate (77 kDa) and the fully glycosylated mature gG-2 (120 kDa) should be identifiable .
Radioimmunoprecipitation:
This technique can detect both the secreted amino-terminal and cell-associated carboxy-terminal portions of gG-2 in virus-infected cell medium .
What molecular mechanisms underlie gG-2 negative HSV-2 clinical isolates?
Research has identified that gG-2 negative HSV-2 clinical isolates result from frameshift mutations:
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Single insertion or deletion of guanine or cytosine nucleotides in the gG-2 gene
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These frameshift mutations result in premature termination codons
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Mutations are typically localized within runs of five or more guanine or cytosine nucleotides
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The mutations can occur at various positions throughout the gene
In one isolate with partially inactivated gG-2, the frameshift mutation was localized upstream of but adjacent to the nucleotides coding for the transmembranous region, resulting in normal production of the secreted portion but a truncated carboxy-terminal portion .
How should primers be designed for complete sequencing of the gG-2 gene?
Research protocols for gG-2 gene sequencing utilize multiple overlapping primers spanning the entire coding region:
| Primer type and position (nt) | Sequence |
|---|---|
| Sense primers | |
| −57–(−39) | 5′ GCACAAAAAGACGCGGCCC |
| 222–240 | 5′ CGTCGTCCGTCACGAGCCC |
| 498–517 | 5′ GCTGGTGCCGATCTGGGACC |
| 847–866 | 5′ TTTATTCGGATGGCACGACC |
| 1052–1071 | 5′ CCTCCGATTCGCCTACGTCC |
| 1372–1392 | 5′ CCCACGTCTACCCACGCGACC |
| 1530–1548 | 5′ CGCCAACGTTTCGGTCGCC |
| 1723–1740 | 5′ GACGACGACAGCGCCACC |
| 1761–1780 | 5′ GAACCCCAACAAACCACCCC |
| Antisense primers | |
| 306–287 | 5′ TGCGCCAAATCCGCGTACC |
| 600–582 | 5′ CTCCCCGCCCACCTCTACC |
| 926–907 | 5′ GGACCGTCATCTAGGGCCCC |
| 1196–1177 | 5′ GTTGCGGCTTGTGTGGCCAT |
| 1430–1413 | 5′ GGAGGGGTTGTTTGGGGCC |
| 1583–1567 | 5′ GCGGTGCCCCGGGTTCC |
| 1780–1761 | 5′ GGGGTGGTTTGTTGGGGTTC |
| 1907–1894 | 5′ TGTTGGGGTGTGGGGCCC |
| 2140–2119 | 5′ TCCCGTCCTTCATCGTTTCTC |
Note: Nucleotides 2842 to 4938 within the HSV-2 HindIII l fragment (encompassing the gG-2 gene/US4) are numbered as 1 to 2097 for reference strain HG52 .
What are the limitations of gG-based serological assays for HSV typing?
Despite gG's utility for type-specific diagnosis, several methodological limitations have been identified:
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Inconsistent results can occur in repeat testing of the same specimen
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Day-to-day fluctuations in assay performance affect reliability
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Protein lot variations may influence weak positive results
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Color intensity variations particularly affect HSV-2 result interpretation
In controlled studies with 100 specimens tested in triplicate:
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For HSV-1: 97 gave consistent results (24 negative, 73 positive)
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For HSV-2: 93 gave consistent results (82 negative, 11 positive)
How do gG-2 gene mutations affect serological detection of HSV-2 infections?
Frameshift mutations in the gG-2 gene can significantly impact serological diagnosis:
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Patients infected with gG-2-negative HSV-2 may lack detectable antibodies against gG-2
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This can lead to false-negative results in type-discriminating serodiagnosis that relies on gG-2 as an antigen
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Some patients with gG-2-negative isolates may still have antibodies against gG-2, suggesting multiple strain infection or reinfection
In a study of five patients with gG-2-negative isolates, seroreactivity varied significantly:
| Patient no. | Endpoint titer to gG-2 antigen | Endpoint titer to gG-1 antigen |
|---|---|---|
| 2434 | 400 | 1,600 |
| 512 | 400 | 100 |
| 453 | - | 800 |
| 147 | - | 100 |
| 4444 | 200 | 200 |
Note: "-" indicates endpoint titer of <100 .
How can researchers account for heterogeneous serological responses in HSV-2 clinical studies?
When designing HSV-2 serological studies, researchers should consider:
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Potential presence of gG-2 negative variants in the study population
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Implementing multiple testing methodologies beyond gG-2-based assays
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Performing repeat testing to identify inconsistent results
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Considering viral isolation and gene sequencing for cases with discrepant serological findings
The presence of antibodies against gG-2 in patients with gG-2-negative isolates suggests potential multiple strain infections or reinfection events. Further studies are needed to clarify whether this represents selection of different viral clones from a heterogeneous primary HSV-2 population or true reinfection with different strains .
What is the research value of gG-2 negative HSV-2 isolates?
gG-2 negative clinical isolates represent valuable research tools for:
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Studying the function of both secreted and mature portions of gG-2
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Investigating essential vs. non-essential viral genes in human infection
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Examining host immune responses to variant viruses
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Testing the reliability of diagnostic assays
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Understanding viral evolution and selection pressures in vivo
These naturally occurring gG-2 deletion mutants provide unique insights that complement laboratory-constructed mutants.
How should experimental design account for gG-2 sequence variability?
When designing experiments involving HSV-2 gG:
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Consider screening clinical isolates for gG-2 expression before selection for experiments
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Use multiple detection methods (immunoblotting, radioimmunoprecipitation) to confirm gG-2 status
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Sequence the complete gG-2 gene using overlapping primers to identify potential mutations
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Test for both secreted and cell-associated portions of gG-2
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Implement controls for known gG-2 positive and negative isolates
The molecular heterogeneity observed in clinical isolates highlights the importance of thorough characterization before experimental use.
What implications do gG-2 negative variants have for vaccine development?
The existence of viable gG-2 negative clinical isolates has significant implications for HSV-2 vaccine development:
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Vaccines targeting only gG-2 may not provide complete protection against all circulating strains
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The rarity of gG-2 negative isolates (0.21% in one study) suggests most wild-type strains maintain gG-2 expression
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Multivalent vaccines targeting multiple viral antigens would provide broader protection
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Understanding the pathogenicity of gG-2 negative strains helps evaluate the importance of including gG-2 in vaccine formulations
Further characterization of these variant strains may provide critical insights for next-generation HSV vaccine design.
Product Science Overview
Herpes Simplex Virus-2 (HSV-2) is a significant human pathogen primarily associated with genital infections. It is a member of the Herpesviridae family, which also includes HSV-1, known for causing orofacial lesions. HSV-2 infections are widespread and can lead to severe health complications, including neonatal herpes, encephalitis, and an increased risk of HIV transmission.
Glycoprotein G (gG) is one of the envelope glycoproteins of HSV-2. It plays a crucial role in the virus’s ability to infect host cells and evade the immune system. The gG protein is unique to HSV-2 and is not found in HSV-1, making it a valuable target for diagnostic assays and vaccine development.
Recombinant gG refers to the glycoprotein G that has been produced using recombinant DNA technology. This involves inserting the gene encoding gG into a suitable expression system, such as bacteria, yeast, or mammalian cells, to produce the protein in large quantities. Recombinant gG is used in various applications, including:
- Diagnostic Assays: Recombinant gG is utilized in serological tests to detect HSV-2-specific antibodies in patient samples. These tests are essential for diagnosing HSV-2 infections, especially in asymptomatic individuals.
- Vaccine Development: The unique properties of gG make it a promising candidate for vaccine development. Researchers are exploring the use of recombinant gG to elicit an immune response that can protect against HSV-2 infection.
- Research: Recombinant gG is used in research to study the structure and function of the glycoprotein, as well as its interactions with the host immune system.
The production of recombinant gG involves several steps:
- Gene Cloning: The gene encoding gG is cloned into an expression vector, which is a DNA molecule used to introduce the gene into the host cells.
- Transformation: The expression vector is introduced into the host cells, which can be bacteria, yeast, or mammalian cells, depending on the desired expression system.
- Protein Expression: The host cells are cultured under conditions that promote the expression of the gG protein.
- Protein Purification: The recombinant gG protein is purified from the host cells using various techniques, such as affinity chromatography, to obtain a high-purity product.
The use of recombinant gG has several significant applications:
- Improved Diagnostics: Recombinant gG-based assays provide high specificity and sensitivity for detecting HSV-2 infections, reducing the likelihood of false-positive results.
- Vaccine Research: The development of a vaccine against HSV-2 is a high priority in the field of infectious diseases. Recombinant gG is a key component in many experimental vaccines, offering hope for effective prevention of HSV-2 infections.
- Understanding Immune Evasion: Studying recombinant gG helps researchers understand how HSV-2 evades the host immune system, leading to better therapeutic strategies.
