Anti-HIV-1 activity of Trim 37

Abstract

Trim 5α was the first member of the tripartite motif (TRIM) family of proteins that was identified to potently restrict human immunodeficiency virus type 1 (HIV-1) replication. The breadth of antiretroviral activity of TRIM family members is an active area of investigation. In this study, we demonstrate that human Trim 37 possesses anti-HIV-1 activity. This antiretroviral activity and the manner in which it was displayed were implicated by (1) decreased viral replication upon Trim 37 transient overexpression in virus-producing cells, (2) correlation of the reduction of viral infectivity with Trim 37 virion incorporation, (3) increased HIV-1 replication during siRNA depletion of Trim 37 expression, and (4) reduction in viral DNA synthesis upon Trim 37 transient overexpression. Our findings provide the first demonstration, to our knowledge, of the potent antiviral activity of human Trim 37, and implicate an antiviral mechanism whereby Trim 37 interferes with viral DNA synthesis.

Fig. 1.

Endogenous levels of Trim 37 in cell lines. Levels of endogenous Trim 37 in the HepG2, 293T and HeLa cell lines and PBMCs. PBMCs were stimulated with phytohaemagglutinin for 72 h followed by treatment with interleukin-2 for an additional 72 h. Cell lysates were prepared and Trim 37 and gamma-tubulin protein expression was analysed by immunoblotting.

Fig. 2.

Trim 37 expression impairs HIV-1 infectivity when expressed in virus-producing cells. 293T cells were co-transfected with an env-minus HIV-1 vector expressing the mouse HSA and GFP, pHIG, and a VSV-G expression plasmid, pCMV-G, along with a Trim 37 expression construct, pTrim37. Forty-eight hours post-transfection, cell culture supernatants were harvested and used to infect HeLa target cells. (a) Viral infectivity was determined by flow cytometry. (b) The graph represents a quantification of the results in (a). (c) RNA extracts were prepared from 293T cells from experiments shown in panel (a) and were analysed by quantitative RT-PCR using Trim 37-specific primers. (d) Comparison of reduction of HIV-1 infectivity when HIV-1 vector virus was produced from 293T cells co-transfected with pHIG, pCMV-G and pTrim37 (10 µg) or from HepG2 cells (which endogenously express high levels of Trim 37) co-transfected with pHIG and pCMV-G and used to infect HeLa target cells. (e) Transient expression of Trim 37 in target cells does not affect HIV-1 infectivity. HeLa target cells were transiently transfected with the Trim 37 expression plasmid (0, 0.5, 5 or 10 µg) for 48 h and then infected with HIV-1 vector virus. Viral infectivity was determined by flow cytometry as described in Methods. All results are presented as the mean±sd from three independent experiments performed in triplicate.

Fig. 3.

siRNA depletion of Trim 37 enhances HIV-1 infectivity. (a) Reduction of Trim 37 expression by Trim 37-directed siRNA. 293T cells were transduced with scrambled or Trim 37-specific siRNA. Twenty-four hours post-transfection, cells were lysed and RNAs extracted for PCR analysis. (b) Increase in HIV-1 infectivity by siRNA depletion of Trim 37. 293T cells were transfected with siRNAs. Twenty-four hours post-transfection, cells were subsequently transfected with pHIG and pCMV-G. Forty-eight hours post-transfection, cell culture supernatants were collected, filtered and used to infect permissive HeLa target cells. The data represent the mean±sd from at least three independent experiments. *Statistical significance (P<0.05).

Fig. 4.

Trim 37 is incorporated into HIV-1 particles. (a) Immunoblot analysis of virus particles. 293T cells were transfected with pHIG and pCMV-G in the presence of the indicated amounts of pTrim37. Forty-eight hours post-transfection, cell culture supernatants were harvested and virus particles were pelleted through a sucrose cushion. The viral particle pellets were resuspended, lysed and subjected to SDS-PAGE. Immunoblot analysis was done using antibodies directed against Trim 37 and p24 Gag. (b) Relative amounts of Trim 37 incorporated into HIV-1 particles. Error bars indicate sd. (c) 293T cells were co-transfected with human codon optimized gag and the expression plasmid for Trim 37. Forty-eight hours post-transfection, cell culture supernatants and cells were harvested. The virus-like particle pellets or cells were resuspended, lysed and subjected to SDS-PAGE. Immunoblot analysis was done using antibodies directed against Gag and Trim 37.

Fig. 5.

Incorporation of Trim 37 impairs viral DNA synthesis. 293T cells were co-transfected with pHIG and pCMV-G alone (control) or with pTrim37. Cell culture supernatants were collected at 48 h post-transfection, filtered and used to infect permissive HeLa target cells. Cells were harvested at 4, 8, 12 and 24 h post-infection for qPCR analysis. (a) Early RT products (i.e. minus-strand SS DNA). (b) LRT products. The data are the mean±sd from experiments done in triplicate.