Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function.

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.

Deciphering the protective role of adaptive immunity to CHIKV/IRES a novel candidate vaccine against Chikungunya in the A129 mouse model.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, recently re-emerged in Africa and spread to islands in the Indian Ocean, the Indian subcontinent, and to South East Asia. Viremic travelers have also imported CHIKV to the Western hemisphere highlighting the importance of CHIKV in public health. In addition to the great burden of arthralgic disease, which can persist for months or years, epidemiologic studies have estimated case-fatality rates of ∼0.1%, principally from neurologic disease in older patients. There are no licensed vaccines or effective therapies to prevent or treat human CHIKV infections. We have developed a live CHIKV vaccine (CHIKV/IRES) that is highly attenuated yet immunogenic in mouse models, and is incapable of replicating in mosquito cells. In this study we sought to decipher the role of adaptive immunity elicited by CHIKV/IRES in protection against wild-type CHIKV infection. A single dose of vaccine effectively activated T cells with an expansion peak on day 10 post immunization and elicited memory CD4(+) and CD8(+) T cells that produced IFN-γ, TNF-α and IL-2 upon restimulation with CHIKV/IRES. Adoptive transfer of CHIKV/IRES-immune CD4(+) or CD8(+) T cells did not confer protection against wtCHIKV-LR challenge. By contrast, passive immunization with anti-CHIKV/IRES immune serum provided protection, and a correlate of a minimum protective neutralizing antibody titer was established. Overall, our findings demonstrate the immunogenic potential of the CHIKV/IRES vaccine and highlight the important role that neutralizing antibodies play in protection against an acute CHIKV infection.

A single amino acid change resulting in loss of fluorescence of eGFP in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus.

Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.

Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

Role of the hypervariable hinge region of phosphoprotein P of vesicular stomatitis virus in viral RNA synthesis and assembly of infectious virus particles.

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase and has multiple functions residing in its different domains. In the present study, we examined the role of the hypervariable hinge region of P protein in viral RNA synthesis and recovery of infectious VSV by using transposon-mediated insertion mutagenesis and deletion mutagenesis. We observed that insertions of 19-amino-acid linker sequences at various positions within this region affected replication and transcription functions of the P protein to various degrees. Interestingly, one insertion mutant was completely defective in both transcription and replication. Using a series of deletion mutants spanning the hinge region of the protein, we observed that amino acid residues 201 through 220 are required for the activity of P protein in both replication and transcription. Neither insertion nor deletion had any effect on the interaction of P protein with N or L proteins. Infectious VSVs with a deletion in the hinge region possessed retarded growth characteristics and exhibited small-plaque morphology. Interestingly, VSV containing one P protein deletion mutant (PDelta7, with amino acids 141 through 200 deleted), which possessed significant levels of replication and transcription activity, could be amplified only by passage in cells expressing the wild-type P protein. We conclude that the hypervariable hinge region of the P protein plays an important role in viral RNA synthesis. Furthermore, our results provide a previously unidentified function for the P protein: it plays a critical role in the assembly of infectious VSV.

The latent membrane protein 1 of Epstein-Barr virus establishes an antiviral state via induction of interferon-stimulated genes.

Epstein-Barr virus (EBV) infection is associated with several human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. In this report, we show that LMP-1 is able to induce the expression of several interferon (IFN)-stimulated genes (ISGs) with antiviral properties such as 2'-5' oligoadenylate synthetase (OAS), stimulated trans-acting factor of 50 kDa (STAF-50), and ISG-15. LMP-1 inhibits vesicular stomatitis virus (VSV) replication at low multiplicity of infection (0.1 pfu/cell). The antiviral effect of LMP-1 is associated with the ability of LMP-1 to induce ISGs; an LMP-1 mutant that cannot induce ISGs fails to induce an antiviral state. High levels of ISGs are expressed in EBV latency cells in which LMP-1 is expressed. EBV latency cells have antiviral activity that inhibits replication of superinfecting VSV. The antiviral activity of LMP-1 is apparently not related to IFN production in our experimental systems. In addition, EBV latency is responsive to viral superinfection: LMP-1 is induced and EBV latency is disrupted by EBV lytic replication during VSV superinfection of EBV latency cells. These data suggest that LMP-1 has antiviral effect, which may be an intrinsic part of EBV latency program to assist the establishment and/or maintenance of EBV latency.

Phosphorylation of vesicular stomatitis virus phosphoprotein P is indispensable for virus growth.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNA-dependent RNA polymerase (RdRp) complex. It is phosphorylated at two different domains. Using defective interfering (DI) RNA or minigenomic RNA templates, we previously demonstrated that phosphorylation within the amino-terminal domain I is essential for transcription, whereas phosphorylation within the carboxy-terminal domain II is necessary for replication. For the present study, we examined the role of the phosphorylation of residues in these domains in the life cycle of VSV. Various mutant P coding sequences were inserted into a full-length cDNA clone of VSV, and the virus recovery, kinetics of growth, and mRNA and protein synthesis were examined. We observed that virus recovery was completely abolished when all three phosphate acceptor sites in domain I or both sites in domain II were replaced with alanine. Single or double mutations in domain I (with the exception of P60/64) or single mutations in domain II had no adverse effect on virus recovery. VSVP227, carrying alanine at position 227, showed reduced kinetics of virus growth but increased kinetics of viral mRNA synthesis in infected cells. More interestingly, this particular virus exhibited a significantly reduced cytopathic effects and apoptosis in infected cells, implying that P may be involved in these processes. Furthermore, we found that DI RNAs of different sizes were generated by high-multiplicity passaging of various mutant VSVs, indicating that the viral RdRp may play a significant role in the process of DI particle generation. Taken together, our results suggest that the phosphorylation of residues in domains I and II of VSV P is indispensable for virus growth.