Type I interferon signaling is required for the APOBEC3/Rfv3-dependent neutralizing antibody response but not innate retrovirus restriction

Abstract

Background: APOBEC3/Rfv3 restricts acute Friend retrovirus (FV) infection and promotes virus-specific neutralizing antibody (NAb) responses. Classical Rfv3 studies utilized FV stocks containing lactate-dehydrogenase elevating virus (LDV), a potent type I interferon inducer. Previously, we showed that APOBEC3 is required for the anti-FV activity of exogenous IFN-alpha treatment. Thus, type I interferon receptor (IFNAR) signaling may be required for the APOBEC3/Rfv3 response.

Results: To test if the APOBEC3/Rfv3 response is dependent on type I IFN signaling, we infected IFNAR knockout versus IFNAR/APOBEC3 double-knockout mice with FV/LDV or LDV-free FV, and evaluated acute FV infection and subsequent NAb titers. We show that LDV co-infection and type I IFN signaling are not required for innate APOBEC3-mediated restriction. By contrast, removal of LDV and/or type I IFN signaling abrogated the APOBEC3-dependent NAb response.

Conclusions: APOBEC3 can restrict retroviruses in a type I IFN-independent manner in vivo. By contrast, the ability of APOBEC3 to promote NAb responses is type I IFN-dependent. These findings reveal novel insights on the interplay between type I IFNs and APOBEC3 in vivo that may have implications for augmenting antiretroviral NAb responses.

Keywords: Deaminase; Friend retrovirus; IFNAR; LDV; Neutralizing antibody.

Fig. 1

APOBEC3/Rfv3 inhibits infectious virus release in an IFNAR KO background. a Experimental design. B6 IFNAR KO and IFNAR/mA3 dKO mice were infected with FV and samples analyzed at the indicated time points. b Infectious viremia was measured by incubating plasma onto susceptible Mus dunni cells for 2 days and determining F-MuLV proviral DNA levels. Infectious viremia was determined for both (left) FV/LDV and (right) LDV-free FV infection. The same samples in (b) were used to determine c plasma viral RNA loads by qPCR. The ratio of the log-transformed infectious titer in (b) and plasma viral loads in (c) were used to estimate d virion infectivity. Each dot corresponds to a mouse and lines correspond to mean values. The total number of mice analyzed was combined from 2 to 3 independent experiments. Data were analyzed using a 2-tailed unpaired Student’s t test, with exact p values shown. Fold-change values in statistically-significant comparisons were based on average non-log-transformed values

Fig. 2

APOBEC3/Rfv3 inhibits acute FV infection of splenocytes independent of type I IFN signaling. Splenocyte FV infection levels were measured by flow cytometry using a glyco-gag specific monoclonal antibody. a Representative flow plots showing glyco-gag+ splenocytes from FV/LDV infected mice at 7 dpi. The percentage of live splenocytes that expressed glyco-gag was evaluated in b FV/LDV and c LDV-free FV infections. Each dot corresponds to a mouse and lines correspond to mean values. The total number of mice analyzed was combined from 2 to 3 independent experiments. Fold-change of mean values per cohort are shown. Data were analyzed using a 2-tailed unpaired Student’s t test, with exact p values shown

Fig. 3

LDV co-infection is critical for the APOBEC3/Rfv3-dependent NAb response. B6 WT and mA3 KO mice were infected with 10⁴ SFFU of a FV/LDV or b LDV-free FV. Plasma samples at 28 dpi were heat-inactivated and the reciprocal plasma dilution that conferred 50% neutralization was computed. Log₄-transformed data are shown and used for statistical analyses. Each dot corresponds to a mouse and lines correspond to mean values. The total number of mice analyzed was combined from 2 independent experiments. Fold-change values in statistically-significant comparisons were based on median non-log-transformed values. Data were analyzed using 2-tailed unpaired Student’s t test, with p values indicated; ns, not significant (p > 0.05)

Fig. 4

APOBEC3/Rfv3-dependent NAb response requires type I IFN signaling. Mice were infected with FV/LDV at two different inoculum doses: a, b 10,000 SFFU and c, d 2000 SFFU. a, c Plasma samples at 28 dpi were heat-inactivated and the reciprocal plasma dilution that conferred 50% neutralization was computed. Log₄-transformed data are shown and used for statistical analyses. b, d FV-specific IgG2b/c titers were determined by endpoint-titration ELISA for mice infected with 10⁴ SFFU of FV/LDV. Native FV virions were coated into ELISA plates and twofold dilutions of plasma were added. IgG2b/c antibodies were detected using a combination of anti-IgG2b and anti-IgG2c conjugates. In all panels, each dot corresponds to a mouse and lines correspond to mean values. The total number of mice analyzed was combined from 2 independent experiments. Data were analyzed using 2-tailed unpaired Student’s t test; ns not significant (p > 0.05)