Identification of rabbit TRIM genes closely related to primate TRIM5. (A) VSV-G-pseudotyped, GFP-encoding vectors derived from the viruses shown were titrated on permissive feline CRFK cells and rabbit SIRC cells. The doses of virus are plotted as multiplicities of infection (MOI) on CRFK cells. Data are representative of three independent experiments. (B) A selection of mammalian TRIM nucleotide sequences was aligned and used to construct a maximum-likelihood phylogenetic tree to consider the relatedness of rabbit TRIM genes to mammalian TRIM sequences. Percent bootstrap support values for 1,000 replicates are shown on the branches. Abbreviations: mac, rhesus macaque; Agm, African green monkey. Branch lengths represent the number of substitutions per site. (C) Alignment of protein sequences of rabbit TRIM5 and rhesus macaque and human TRIM5α reveals strong conservation between RING and B-Box 2 (BB) domains and the beta sheet sequences at the core of the B30.2 domain (arrows) (23). The coiled-coil (CC) domain is also shown. Dotted lines mark variable regions V1 to V4. HuTRIM5, human TRIM5; rbTRIM5, rabbit TRIM5; asterisk, identical residue; colon, conserved substitution; period, semiconserved substitution; gap, no conservation.

Expression of rabbit TRIM5 (rbTRIM5) in permissive feline cells confers the ability to restrict retroviral infection. CRFK cells were transduced with MLV retroviral vector encoding rabbit TRIM5 and dsRED express and cloned by dilution. They were then tested for their permissivity to VSV-G-pseudotyped, GFP-encoding retroviral vectors as shown. The data plotted are the titers of these viruses on CRFK cells expressing rabbit TRIM5 versus those on unmodified CRFK cells (U) as a control. Errors are standard deviations for titers determined at three different multiplicities of infection. Data shown are representative of experiments performed on two independent clones of cells.

The viral determinant for restriction of HIV-1 by rabbit TRIM5 (rbTRIM5) is the CA. (A) Cartoons of HIV-1 and SIVmac gag are shown to illustrate the structure of the HIV-1/SIVmac SHIV hybrids used. H/SCA is HIV-1 with CA amino acids 1 to 146 from SIVmac. SHIV3+ is SIVmac with the CA-p2 domains of HIV-1. MA, matrix; NC, nucleocapsid. (B) Absolute titers of wild-type and SHIV vectors were determined on CRFK cells expressing rabbit TRIM5 (white bars) and unmodified CRFK cells (U; black bars) as a control. (C) Titers of the wild-type and SHIV vectors on rabbit SIRC cells. Doses are standardized on permissive feline CRFK cells. Errors are standard deviations for titers determined at different doses and are representative of experiments performed on two independent clones.

Introduction of two independent shRNA sequences directed against rabbit TRIM5 into SIRC cells increases their permissivity to restricted retroviruses. SIRC cells were transduced with a high multiplicity of infection (>2) with the MLV pSIREN RetroQ vectors, each bearing one of two independently designed shRNAs (sh1 and sh2) directed against rabbit TRIM5 or shRNA targeting luciferase (shLuc) as a control. Forty-eight hours later, the permissivity of these cells, as well as unmodified SIRC cells (U), was tested by titrating serial dilutions of VSV-G-pseudotyped, GFP-encoding vectors derived from the viruses shown. The percentages of infected cells were measured by FACS 48 h after GFP-encoding vector infection, and data are plotted as numbers of infectious units/ml. Errors are standard deviations for titers determined at three different multiplicities and are representative of data from two independent experiments.

Rabbit TRIM5 (rbTRIM5) blocks DNA synthesis by restricted virus. CRFK cells expressing TRIM5 were infected in triplicate with VSV-G-pseudotyped, GFP-encoding vectors derived from the viruses shown. (A) Two samples were extracted for DNA and viral DNA measured by QPCR using primers/probe specific to GFP. Data plotted are the numbers of viral DNA molecules per 100 ng of total DNA after infection of unmodified CRFK cells (U; black bars) or CRFK cells expressing rabbit TRIM5 (gray bars). (B) The third sample was subjected to FACS 48 h after infection to determine the percentage of infected cells. Results for negative-control infections of unmodified cells with boiled virus are shown (white bars or arrows for values below the limit of reliable detection). Errors are standard deviations for duplicate samples and are representative of experiments performed on two independent clones.

Human TRIM5 isoforms are dominant negatives against restriction of retroviruses in SIRC cells. Rabbit SIRC cells were left uninfected (U; black bars) or infected with empty vector (EV; dark gray bars), MLV vector encoding human TRIM5α (light gray bars), or human TRIM5δ (white bars). Forty-eight hours later, the cells were challenged with GFP-encoding SIVmac, FIV, HIV-1, or HIV-2. The increases (n-fold) in infectivity of the GFP-encoding virus are plotted. Errors are standard deviations for parallel infections. Results are representative of two independent experiments performed with independent virus stocks.