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Mate-conserved rhTRIM5 epitope and an actin antibody as a cell lysate
Mate-conserved rhTRIM5 epitope and an actin antibody as a cell lysate loading control. The results (Figure 1A) showed that, in addition to the endogenous human TRIM5 band at 56 kDa (present in the untransduced JR5 cell lysate), there were bands at 59 and 57 kDa in the hAgmT and hgorT lysates, respectively, corresponding to the expected molecular masses (TRIM5 with the HA-tag) of the hAgmTRIM5 and hgorTRIM5 proteins. Similarly, the AgmT cell lysates contained bands at 56 kDa and 58 kDa, consistent with human and AgmTRIM5 proteins, respectively. In contrast, the gorT line contained only one band at 56 kDa, yet with a greater intensity relative to the bands in the other samples (Figure 1A). Due to their nearly identical molecular mass, ectopic gorilla and endogenous human TRIM5 proteins co-migrate. Measurement of the fluorescence intensities of both the xenogeneic and endogenous TRIM5 bands and normalization by actin band signal revealed that the range of ectopic TRIM5 expression was close to normal physiological levels (Figure 1B), only 1- to 2-fold over that of endogenous human TRIM5 among the different transduced cell lines.Xenogeneic TRIM5 expression modestly restricts HIV-1 and SIV single-round infectionGFP fluorescence revealed that AgmT cells exhibited somewhat PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27107493 more resistance to HIV vector transduction than the gorT PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28154141 cells, with a A-836339 web 4-fold compared to a 3-fold resistance to infection, respectively (Figure 1C). These results generally agree with prior published studies. To examine infection in a more physiological system, the xenogeneic TRIM5-expressing cells were infected with either HIV-1 or SIVmac239 and Gag-positive cells were measured 40-h post-infection by intracellular flow cytometry with CA antibodies. To control for non-specific virion binding, the numbers of Gag-positive cells were adjusted for the background present in the heatinactivated virus. The results were similar to those of the vector-derived data: the infection of AgmT cells was 90 lower for both viruses, and gorT cells demonstrated a more modest reduction of infection, approximately 70 (Figure 1D). For comparison to the more extensively studied rhesus macaque system, we examined the effect of xenogeneic rhTRIM5 in our short-term infection assay. JR5 were transduced with the Babe-rhT vector, which expresses rhTRIM5 and puromycin acetyltransferase, and selected with puromycin to produce the rhT cell line. Immunoblot analysis of rhT lysates revealed only a single, more intense TRIM5-sized band due to the close molecular masses of rhTRIM5 and its human counterpart (Figure 1A). Measurement of the bands found a 6-fold increase in the TRIM5 over that of the JR5 controls (Figure 1B), a higher level of exogenous expression that the other JR5/TRIM5 cell lines. Consistent with prior results, the rhTRIM5-expressing cells reduced HIV-1 infectivity by an amount similar to that of the AgmTRIM5 proteins in the short term assay and did not significantly affect SIV infection, as expected (Figure 1D). Modest restriction was also observed by assaying HIV-1 and SIVmac239 in Agm- and gorTRIM5-expressing TZMbl cells (data not shown). Thus, in our systems, xenogeneic expression of these three TRIM5 proteins results in a modest level of restriction in single-round/ short-term assays and recapitulates most prior studies of Agm- and rhTRIM5 [9,18,21-24,26-30].Strong AgmTRIM5 replication restriction after a cell-free challengeThe magnitude of TRIM5 restriction has been well established by c.

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