Primate TRIM34 is a broadly-acting, TRIM5-dependent lentiviral restriction factor

Abstract

Human immunodeficiency virus (HIV) and other lentiviruses adapt to new hosts by evolving to evade host-specific innate immune proteins that differ in sequence and often viral recognition between host species. Understanding how these host antiviral proteins, called restriction factors, constrain lentivirus replication and transmission is key to understanding the emergence of pandemic viruses like HIV-1. Human TRIM34, a paralogue of the well-characterized lentiviral restriction factor TRIM5α, was previously identified by our lab via CRISPR-Cas9 screening as a restriction factor of certain HIV and SIV capsids. Here, we show that diverse primate TRIM34 orthologues from non-human primates can restrict a range of Simian Immunodeficiency Virus (SIV) capsids including SIV _AGM-SAB , SIV _AGM-TAN and SIV _MAC capsids, which infect sabaeus monkeys, tantalus monkeys, and rhesus macaques, respectively. All primate TRIM34 orthologues tested, regardless of species of origin, were able to restrict this same subset of viral capsids. However, in all cases, this restriction also required the presence of TRIM5α. We demonstrate that TRIM5α is necessary, but not sufficient, for restriction of these capsids, and that human TRIM5α functionally interacts with TRIM34 from different species. Finally, we find that both the TRIM5α SPRY v1 loop and the TRIM34 SPRY domain are essential for TRIM34-mediated restriction. These data support a model in which TRIM34 is a broadly-conserved primate lentiviral restriction factor that acts in tandem with TRIM5α, such that together, these proteins can restrict capsids that neither can restrict alone.

Figure 1.. Diverse primate TRIM34 orthologues restrict SIV_AGM-SAB, SIV_AGM-SAB, and SIV_MAC capsids in the presence of TRIM5α

a. THP-1 TRIM34 clonal KO cells were generated by electroporation of multiplexed sgRNA against TRIM34 and single cell sorting (ICE KO score = 96%). THP-1 TRIM34 KO cells were transduced with doxycycline-inducible HA-tagged primate TRIM34 orthologues or empty vector control. Primate TRIM34 expression was induced with doxycline. Expression levels were visualized by immunoblotting 72 h post-induction. b-f. Primate TRIM34 expression was induced in THP-1 TRIM34 clonal KO cells. 1 day post-induction, cells were either challenged with chimeric virus particles containing HIV-1 capsids co-expressing zsGreen (b) or SIV capsids co-expressing eGFP including SIV_CPZ (c), SIV_AGM-SAB (d), or SIV_MAC (f); or challenged with VSV-G pseudotyped SIV_AGM-TAN-luc (e). Infection was quantified 2 dpi by flow cytometry (b-d, f) or luminometry (e). Relative infectivity in induced cells (white bars) is normalized to average infectivity in uninduced control cells (grey bars). Data are represented as mean +/− s.d. of 3 technical replicates from 1 representative experiment. One-sided p values were calculated by Welch’s t-test. ns = nonsignificant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 2.. TRIM34 requires TRIM5α to restrict SIV_AGM-SAB.

a-b. THP-1 TRIM34 clonal KO cells containing doxycycline-inducible TRIM34 from humans (a) or rhesus macaques (b) were transduced with lentiviral vectors encoding Cas9 and 1 of 2 independent sgRNA against TRIM5 or a non-targeting control to generate pooled TRIM5 knockout or NTC cell lines. KO efficiency was assessed by ICE analysis. For human TRIM34-expressing cell lines, ICE KO efficiency scores were as follows: TRIM5 sgRNA 1 (squares) = 76, TRIM5 sgRNA 2 (circles) = 90. For rhesus macaque TRIM34-expressing cells, ICE KO scores were as follows: TRIM5 sgRNA 1 (squares) = 83, TRIM5 sgRNA 2 (circles) = 93. Thus, there existed 4 different cellular conditions: no TRIM34 or TRIM5α expression = black symbols, only endogenous TRIM5α expression = blue symbols, only overexpressed TRIM34 = green symbols, both endogenous TRIM5α and overexpressed TRIM34 = red symbols. 1 day after doxycycline induction, cells were infected with SIV_AGM-SAB CA particles; each infection was performed in triplicate. 2 dpi, infectivity was quantified by flow cytometry. Relative infectivity is normalized to mean infectivity in TRIM5 KO, TRIM34-uninduced cells (black symbols). Data are represented as mean +/− s.d. from 1 representative experiment. Mean represents average across all 6 replicates. c. HeLa cells were transduced to stably express primate TRIMΔSPRY orthologues or empty vector control. Expression was visualized by immunoblotting. d-e. HeLa cells stably expressing primate TRIM34ΔSPRY orthologues or empty vector control were infected with N-MLV (d) or NB-MLV (e). Level of infection was quantified 2 dpi by flow cytometry. Data are represented as mean +/− s.d. from 1 representative experiment. Infections were performed in triplicate. a-b, d-e. p values were calculated by Brown-Forsythe and Welch’s 1-way ANOVA with Dunnett’s T3 test for multiple comparisons. ns = nonsignificant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 3.. TRIM5α v1 loop is necessary for restriction of SIV_AGM-SAB.

a. THP-1 TRIM34 clonal KO cells were electroporated with multiplexed sgRNA against TRIM5. Single cell clonal lines were generated by limiting dilution to generate a THP-1 TRIM34 TRIM5 double KO clonal cell line. Cells were doubly transduced with doxycycline-inducible, HA-tagged human TRIM34 or empty vector control in tandem with doxycycline-inducible, FLAG-tagged human TRIM5α, TRIM5Δv1, TRIM5 R332P, or empty vector control. Expression levels were visualized by immunoblotting 72 h post-induction. b-d. THP-1 TRIM34 TRIM5 double KO clonal cells co-expressing doxycycline-inducible human TRIM34 or empty vector control and human TRIM5α (grey bars), TRIM5Δv1 (red bars), TRIM5 R332P (blue bars), or empty vector control were infected with SIV_AGM-SAB (b), HIV-1 N74D (c), or HIV-1 (d) CA 1 day post-induction. Levels of infection were quantified 2 dpi by flow cytometry. Relative infectivity in induced cells (solid bars) is normalized to average infectivity in uninduced control cells (not shown). Data are represented as mean +/− s.d. of 3 technical replicates from 1 representative experiment. One-sided p values were calculated by Welch’s t-test. ns = nonsignificant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 4.. TRIM34 SPRY domain is necessary for restriction of SIV_AGM-SAB.

a. Schematic of TRIM34 RBCC-TRIM5 SPRY chimera b. THP-1 TRIM34 clonal KO cells were generated by electroporation of multiplexed sgRNA against TRIM34 and single cell sorting. Cells were transduced with doxycycline-inducible HA-tagged human TRIM34, TRIM5α, or TRIM34-TRIM5α chimeras. Expression levels were visualized by immunoblotting 72 h post-induction. c. THP-1 TRIM34 clonal KO cells expressing doxycycline-inducible human TRIM34, TRIM5α, or TRIM34 RBCC-TRIM5 SPRY were infected with SIV_AGM-SAB CA particles 1 day post-induction. Level of infection was quantified 2 dpi by flow cytometry. Relative infectivity in induced cells (white bars) is normalized to average infectivity in uninduced control cells (grey bars). Data are represented as mean +/− s.d. from 1 representative experiment comprising 3 technical replicates. One-sided p values were calculated by Welch’s t-test. ns = nonsignificant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.