Independent evolution of an antiviral TRIMCyp in rhesus macaques

Abstract

The antiretroviral restriction factor TRIM5 has recently emerged as an important mediator of innate immunity and species-specific inhibition of retroviral replication in mammals. Selection pressure from pathogenic infection has driven rapid evolution of TRIM5 genes, leading to the antiviral specificities we see today. Remarkably, the New World owl monkey (Aotus trivirgatus) encodes a TRIM5 protein in which the antiviral determinants in the B30.2 domain have been replaced by cyclophilin A (CypA) encoded by a retrotransposed cDNA. The owl monkey TRIMCyp protein restricts infection by a subset of lentiviruses that recruit CypA to their capsids, including HIV-1 and feline immunodeficiency virus. Here, we show that the Old World monkey, rhesus macaque (Macaca mulatta), also encodes a TRIMCyp protein that has arisen independently from that in owl monkeys. The rhesus TRIMCyp is encoded by a single, but common, allele (Mamu7) of the rhesus TRIM5 gene, among at least six further alleles that encode full-length TRIM5 proteins with no homology to CypA. The antiviral specificity of the rhesus TRIMCyp is distinct, restricting infection of HIV-2 and feline immunodeficiency virus but not HIV-1. Restriction by rhesus TRIMCyp is before reverse transcription and inhibited by blocking CypA binding, with cyclosporine A, or by mutation of the capsid CypA binding site. These observations suggest a mechanism of restriction that is conserved between TRIMCyp proteins. The lack of activity against HIV-1 suggests that Mamu7 homozygous animals will be null for TRIM5-mediated restriction of HIV-1 and could contribute to improved animal models for HIV/AIDS.

Fig. 1.

Identification of a rhesus TRIMCyp. (A) Diagram of the TRIM5α protein showing polymorphisms between proteins encoded by Mamu1 and Mamu7. The position of nonsynonymous (above) and synonymous differences (below) are shown. (B) The sixth intron of Mamu7 bears a mutation at the splice acceptor site (star). Exon7 sequence is shown (boxed). Conserved nucleotides are indicated (asterisk). (C) Alignment of the protein sequences of rhesus TRIMCyp (Mamu7) and owl monkey TRIMCyp (TCyp). Asterisk, identical residue; colon, conserved substitution; period, semiconserved substitution; gap, no conservation. RING, Bbox2 (BB), coiled coil (CC), and CypA domains are shown. (D) Diagram indicating splicing of TRIM5α encoded by Mamu1, rhesus TRIMCyp (Mamu7), and owl monkey TRIMCyp. Noncoding (gray) and coding (black) exons and CypA (striped) sequences are shown. In owl monkey, TRIMCyp CypA is encoded by a CypA cDNA in the seventh intron (20). (E) Genomic sequence of the Mamu7 CypA sequence. Target site duplication (TSD), splice acceptor (asterisk), start codon (circle), and polyadenylation signal and poly(A) sequences (underlined), which are typical of L1-mediated retrotransposition, are shown. (F) PCR using primers on either side of the CypA insertion indicates that it is unique to the Mamu7 allele. Mamu genotypes, as determined by sequencing the exon eight PCR product, are shown. H₂O denotes water control.

Fig. 2.

Rhesus TRIMCyp restricts HIV-2 and FIV but not HIV-1, SIVmac, EIAV, or MLV. (A–G) Infectious titers of the viruses indicated were determined on unmodified feline cells (CRFK), CRFK cells bearing empty vector (EXN), CRFK cells expressing TRIM5α (Mamu1), rhesus (Mamu7), or owl monkey TRIMCyp (OMTC). Errors are standard deviations of titers determined at three multiplicities of infection and are representative of experiments performed with independent viral stocks. (H) Expression levels of rhesus TRIM5α (Mamu1), rhTRIMCyp (Mamu7), or owl monkey TRIMCyp (OMTC) were compared in CRFK cells or CRFK cells bearing empty vector (EXN) by Western blot analysis for the HA tag. β-actin was detected on parallel blots as a loading control.

Fig. 3.

Overexpression of rhesus TRIMCyp inhibits restriction by rhesus TRIM5α. FRhK4 cells were transduced with equal doses of MLV vector encoding TRIM5α or rhesus TRIMCyp. A human TRIM34 expression vector was used as a positive control. GFP-encoding VSV-G pseudotypes of HIV-1 (A), HIV-2 (B), or MLV-B (C) were titrated on the modified cells 48 h later. Titers are expressed as infectious units/ml (IU/ml) determined as described (Fig. 2). Errors are standard deviations derived from triplicate experiments and are representative of at least two experiments using independent viral stocks. (D) Parallel infections were harvested at 48 h, and TRIM protein expression was determined by Western blotting analysis for the N-terminal HA tag.

Fig. 4.

Drugs or mutations that inhibit CypA recruitment to capsid prevent rhTRIMCyp antiviral activity. Titers of wild-type HIV-2 (A), FIV (B), or HIV-2 bearing capsid mutation G87A (C) were measured on CRFK cells (CRFK), CRFK cells bearing empty vector (EXN), CRFK cells expressing TRIM5α (Mamu1), CRFK cells expressing rhesus TRIMCyp (Mamu7), or CRFK cells expressing owl monkey TRIMCyp (OMTC) in the presence (black bars) or absence (gray bars) of 5 μM CSA. Errors are standard deviations of titers determined at three multiplicities of infection and are representative of experiments performed with independent viral stocks.

Fig. 5.

Rhesus TRIMCyp blocks restricted viral reverse transcription in a proteasome-dependent way. Cells expressing rhTRIMCyp (Mamu7), or unmodified cells (CRFK), were infected with HIV-2 (A and B) or FIV (C and D) in triplicate in the presence (black bars) or absence (gray bars) of proteasome inhibitor. One sample was subjected to FACS to determine infectious titer at 48 h (A and C), and total DNA was purified from the second and third sample 6 h after infection for measurement of viral DNA synthesis (B and D). Viral DNA synthesis is expressed as molecules of GFP template per 100 ng of total DNA. Errors are standard deviations of values derived from parallel infections, and data are representative of independent experiments performed by using independent stocks of virus.