Noninfectious retrovirus particles drive the APOBEC3/Rfv3 dependent neutralizing antibody response

Abstract

Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV) infection by promoting a more potent neutralizing antibody (NAb) response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.

Figure 1. B6 mA3 promotes noninfectious particle release during acute FV infection.

(B6×BALB/c)F₁ mice were infected with 140 SFFU of FV complex and 7 dpi plasma samples from B6 mA3 ⁺ and B6 mA3 ⁻ F₁ mice were subjected to infectious viremia titration in Mus dunni cells and viral RNA copy determinations by quantitative PCR. Virion infectivity for each sample was measured by taking the ratio of infectious titer and viral load. (A) Infectious viremia and (B) plasma viral load during acute infection of (B6×BALB/c)F₁ mice are shown as log₁₀ values. (C) Virion infectivity was calculated by taking the ratio of log₁₀ infectious titer and plasma viral load. (D) Virion infectivity was also compared with non-log transformed values, setting the average infectious titer per viral copy number of (B6 mA3 ^−/−×BALB/c)F₁ as 100%. Samples below the assay limit of detection (below the dotted lines in panel A) were excluded in this calculation. Solid lines correspond to mean values, and p values from a two-tailed Student's t test are shown. Each dot corresponds to an infected mouse. Error bars correspond to the standard error of the mean.

Figure 2. Mechanism of B6 mA3 inhibition of plasma virion infectivity.

(A) General strategy to investigate FV evolution in vivo. FV env sequences from the inoculum stock, 7 dpi plasma, and reverse transcripts following infection of target Mus dunni cells with 7 dpi plasma were compared with each other. (B) Sequence characterization of the FV inoculum quasispecies. (Left) Contemporary FV sequences (gray circles) cluster in phylogenetic analyses with >80% bootstrap value (asterisk), and diverge from a 1983 sequence, FB29. (Right) Sequence alignment against the consensus showed 13 variant sites (vertical bars). These variations were excluded from subsequent analyses of FV mutation rates in infected mice. The two most divergent FV sequences differed by 0.6% in nucleotide identity. (C) Virion RNA mutational loads from (B6×BALB/c)F₁ mice (140 SFFU) 7 dpi plasma were compared against the FV inoculum consensus. No significant difference in mutation rates was observed (Chi-Square test). (D) Reverse transcripts following infection of Mus dunni cells with plasma virions were amplified using conditions to bias for G-to-A mutational detection. Substitution rates were tabulated from a combined consensus sequence derived from the FV inoculum (panel B) and the corresponding plasma virions (panel C). mA3-associated G-to-A substitutions is associated with (B6 mA3 ^+/+×BALB/c)F₁ status by Chi-Square test. (E) mA3 inhibits FV early reverse transcription. DNA from Mus dunni cells infected for 2 days with plasma virions were subjected to quantitative PCR for early (R-U5) and late (R-gag) reverse transcripts, and normalized to beta-actin copy number and input virus. Differences in means were analyzed using a two-tailed Student's t-test.

Figure 3. B6 mA3 is a saturable innate restriction factor in vivo.

(A) Saturability of innate mA3 restriction. Cohorts of (B6 mA3^+/+×BALB/c)F₁ and (B6 mA3 ^−/−×BALB/c)F₁ mice were infected with varying doses of FV and infectious viremia in 7 dpi plasma samples were measured. The fold-difference in mean 7 dpi viremia between the wild-type and mA3-deficient F₁ mice for each dose is shown. Dashed lines correspond to the limit of detection of the focal infectivity assay (600 FFU/ml). (B) Noninfectious FV release despite decreased mA3 restriction. Plasma viral loads (Left) and virion infectivity (right) were determined for mice infected with 500 SFFU of FV. Plasma virion infectivity was significantly higher in (B6 mA3^+/+×BALB/c)F₁ compared to (B6 mA3 ^−/−×BALB/c)F₁ mice. A similar result was observed with a lower infection dose (140 SFFU; Figure 1C). Each dot corresponds to an infected mouse. Differences in means were analyzed using a two-tailed Student's t-test.

Figure 4. NAb responses in (B6×BALB/c)F₁ mice.

(A) Plasma samples at 28 dpi from (B6 mA3^+/+×BALB/c)F₁ and (B6 mA3 ^−/−×BALB/c)F₁ mice were heat-inactivated and analyzed for neutralization potency and virion binding. (B) B6 mA3 influences NAb responses in (B6×BALB/c)F₁ mice. (Left) Neutralization curves were plotted (mean values are shown), and used to (Right) interpolate 50% inhibitory concentration (IC₅₀) values. (C) B6 mA3 dependent NAb responses correlate with IgG antibodies directed against native virions. Endpoint ELISAs were performed on individual plasma samples against virions that were not treated (native) or treated (detergent-lysed) with 1% Empigen-BB detergent. Values correspond to log₂-transformed reciprocal plasma dilutions that corresponded to a cut-off based on 2× mean background absorbance. Each dot corresponds to an infected mouse. Differences in means were analyzed using a two-tailed Student's t-test.

Figure 5. Model for mA3 action and FV-specific humoral immunity.

In Rfv3 resistant mice, high endogenous levels of mA3 may overwhelm putative FV-encoded antagonists such as Glyco-Gag, resulting in functional mA3 incorporated into budding virus particles. In contrast, low levels of mA3 in Rfv3 susceptible strains could efficiently be inactivated. In both strains, similar levels of virus particles released during acute infection could facilitate antigen-specific B cell development. However, virions derived from Rfv3 resistant strains encounter an early post-entry reverse transcription block in target cells. Thus, antibody affinity maturation can occur against functional envelope trimers with decreased pathology in Rfv3 resistant mice. In contrast, fully infectious virions from Rfv3 susceptible strains could directly infect B cells (and other immune cells), resulting in immune dysfunction and weaker development of NAbs. We hypothesize a similar scenario during acute HIV-1 infection, except that: (1) the antagonist is HIV-1 Vif, which degrades hA3G/hA3F; and (2) the targets are CD4⁺ T cells, which in germinal centers are in direct contact with and provide help for antigen-specific B cell development.