Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs.

Alphaviruses are positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV), the prototype alphavirus, preferentially infects neurons in mice and is a model system for studying mechanisms of viral clearance from the nervous system. Antibody specific to the SINV E2 glycoprotein plays an important role in SINV clearance, and this effect is reproduced in cultures of infected mature neurons. To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons. Differentiated AP-7 rat olfactory neuronal cells, an in vitro model for mature neurons, were infected with SINV and treated with anti-E2 antibody. Whole-cell RNA lysates were collected for sequencing of poly(A)-selected RNA 24, 48, and 72 h after infection. Three primary species of viral RNA were produced: genomic, subgenomic, and defective viral genomes (DVGs) encoding the RNA capping protein nsP1. Antibody treatment resulted in overall lower production of SINV RNA, decreased synthesis of subgenomic RNA relative to genomic RNA, and suppressed production of the nsP1 DVG. The nsP1 DVG was packaged into virus particles and could be translated. Because antibody-treated cells released a higher proportion of virions with noncapped genomes and transient transfection to express the nsP1 DVG improved viral RNA capping in antibody-treated cells, we postulate that one mechanism by which antibody inhibits SINV replication in neurons is to suppress DVG synthesis and thus decrease production of infectious virions containing capped genomes. IMPORTANCE Alphaviruses are important causes of viral encephalomyelitis without approved treatments or vaccines. Antibody to the Sindbis virus (SINV) E2 glycoprotein is required for immune-mediated noncytolytic virus clearance from neurons. We used direct RNA nanopore sequencing to evaluate how anti-E2 antibody affects SINV replication at the RNA level. Antibody altered the viral RNAs produced by decreasing the proportion of subgenomic relative to genomic RNA and suppressing production of a previously unrecognized defective viral genome (DVG) encoding nsP1, the viral RNA capping enzyme. Antibody-treated neurons released a lower proportion of SINV particles with capped genomes necessary for translation and infection. Decreased nsP1 DVG production in antibody-treated neurons led to lower expression of nsP1 protein, decreased genome capping efficiency, and release of fewer infectious virus particles. Capping was increased with exogenous expression of the nsP1 DVG. These studies identify a novel alphavirus DVG function and new mechanism for antibody-mediated control of virus replication.

Development of encoded Broccoli RNA aptamers for live cell imaging of alphavirus genomic and subgenomic RNAs.

Sindbis virus (SINV) can infect neurons and cause encephalomyelitis in mice. Nonstructural proteins are translated from genomic RNA and structural proteins from subgenomic RNA. While visualization of viral proteins in living cells is well developed, imaging of viral RNAs has been challenging. RNA aptamers that bind and activate conditional fluorophores provide a tool for RNA visualization. We incorporated cassettes of two F30-scaffolded dimers of the Broccoli aptamer into a SINV cDNA clone using sites in nsP3 (genomic RNA), the 3'UTR (genomic and subgenomic RNAs) and after a second subgenomic promoter resulting in 4-28 Broccoli copies. After addition of the cell-permeable 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI-1T) conditional fluorophore and laser excitation, infected cells emitted green fluorescence that correlated with Broccoli copy numbers. All recombinant viruses replicated well in BHK and undifferentiated neural cells but viruses with 14 or more Broccoli copies were attenuated in differentiated neurons and mice. The signal survived fixation and allowed visualization of viral RNAs in differentiated neurons and mouse brain, as well as BHK cells. Subgenomic RNA was diffusely distributed in the cytoplasm with genomic RNA also in perinuclear vesicle-like structures near envelope glycoproteins or mitochondria. Broccoli aptamer-tagging provides a valuable tool for live cell imaging of viral RNA.

Visualization of cell-type dependent effects of anti-E2 antibody and interferon-gamma treatments on localization and expression of Broccoli aptamer-tagged alphavirus RNAs.

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7-8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.

Development and characterization of Sindbis virus with encoded fluorescent RNA aptamer Spinach2 for imaging of replication and immune-mediated changes in intracellular viral RNA.

Viral RNA studies often rely on in situ hybridization and reverse transcriptase-PCR to provide snapshots of RNA dynamics in infected cells. To facilitate analysis of cellular RNAs, aptamers Spinach and Spinach2 that bind and activate the conditional fluorophore 3, 5-difluoro-4-hydroxybenzylidene imidazolinon have been developed. To determine the feasibility of applying this technology to viral RNA, we have used cDNA clones of the TE strain of Sindbis virus (SINV) to construct multiple viruses containing one or two copies of tRNA-scaffolded Spinach2 after a second subgenomic promoter, TEds-1Sp and TEds-2Sp within the 3'UTR, TE-1UTRSp, or after a second subgenomic promoter and in the 3'UTR, TEds-1Sp+1 UTRSp. TEds-1Sp+1 UTRSp gave the brightest signal and replicated well in cell culture, while TEds-2Sp was the dimmest and replicated poorly. Selection of baby hamster kidney cells infected with TEds-1Sp+1 UTRSp for improved signal intensity identified a virus with a stronger signal and point mutations in the tRNA scaffold. Imaging of SINV in BHK cells showed RNA to be concentrated in filopodia that contacted and transferred RNA to adjacent cells. The effect of treatment with anti-E2 antibody, which effects non-cytolytic clearance of SINV from neurons, on viral RNA was cell-type-dependent. In antibody-treated BHK cells, intracellular viral RNA increased and spread of infection continued. In undifferentiated and differentiated AP7 neuronal cells antibody treatment induced viral RNA clearance. Both viruses with two inserted aptamers were prone to deletion. These studies form the basis for further development of aptamer-labelled viral RNAs that will facilitate functional studies on the dynamics of infection and clearance.

Vaccination of rhesus macaques with a recombinant measles virus expressing interleukin-12 alters humoral and cellular immune responses.

Lack of a vaccine for infants and immunosuppression after infection are problems associated with measles virus (MV). Because interleukin (IL)-12 has been used successfully as a vaccine adjuvant and because inhibition of IL-12 expression has been associated with immunosuppression during measles, the addition of IL-12 may enhance the immune response to MV. To determine the effect of IL-12 supplementation, rhesus macaques were vaccinated with a recombinant MV expressing IL-12; these macaques had increased interferon-gamma production by CD4(+) T cells, decreased production of IL-4, and lower levels of MV-specific immunoglobulin G4 and neutralizing antibody. Lymphoproliferative responses to mitogen were not improved. IL-12 supplementation altered the T helper type 2 bias of the immune response after MV vaccination, had a detrimental effect on the protective neutralizing antibody response, and did not improve other manifestations of immunosuppression. Reduced IL-12 levels are not the sole factor in MV-induced immunosuppression.

Measles virus infection of rhesus macaques affects neutrophil expression of IL-12 and IL-10.

Cytokine production by phagocytic cells is an important component of the immune response to infection with a variety of pathogens. Neutrophils are phagocytic cells capable of expressing interleukin (IL)-12 and IL-10 in response to infection with parasites, fungi, and bacteria. These cytokines are postulated to play important roles in the immune response during measles. Neutrophils isolated from naive or measles virus (MV)-infected rhesus macaques expressed IL-12 and IL-10 following in vitro stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Stimulated neutrophils secreted proportionally more of the biologically active IL-12 heterodimer p70 and more IL-10 than similarly stimulated peripheral blood mononuclear cells (PBMCs). During MV infection, the number of IL-12 and IL-10-producing neutrophils and monocytes initially decreased. Subsequently, IL-12 levels were restored, and IL-10 expression rose above baseline in both cell types. Increased IL-10 production by neutrophils and monocytes during MV infection may play a role in measles-induced immunosuppression.