TSG101 is a housekeeping protein and has been implicated in a number of cellular functions, including mitotic spindle formation, genome stability and endosomal sorting [40] . Essential in endosomal sorting, TSG101 interacts directly with ubiquitinated proteins and internalizes them into the multivesicular body pathway for degradation. During replication, viruses require a retrograde movement from the cell interior to the outer membrane [40] . A number of reports have shown that TSG101 is involved in the virus fusion/budding process from the cellular membrane. These viruses include human immunodeficiency virus (HIV) and hepatitis C virus (HCV) [40] [41] [42] . Recently, a tentative TSG101-targeting peptide, FGI-104, was shown to have a broad-spectrum ability to inhibit infections by several viruses including HIV, HCV and PRRSV [43] . However, there is no direct mechanistic evidence that has demonstrated the involvement of TSG101 in the control of PRRSV replication. To study this possibility, we produced MARC-145 cell lines with targeted suppression of endogenous TSG101 expression. MARC-145 cells were transfected with a pGFP-V-RS vector expressing a 29 nt shRNA (OriGene, Rockville, MD, USA) against a conserved region of TSG101. Two puromycin-resistant colonies showing significant suppression of TSG101 at both RNA and protein levels, were selected. As shown in Figure 2A , cells from colony 1 had less than 10% endogenous expression of tsg101 RNA, and those from colony 9 about 20% of endogenous expression of tsg101 RNA, as well as 70-80% reduction in TSG101 protein. We then compared PRRSV replication in these TSG101-suppressed cells with control MARC-145 cells or cells transfected with scrambled shRNA constructs. Using either GFP-or DsRed-expressing PRRSV, the retarded replication of viruses in TSG101-suppressed cells was demonstrated between 12 and 48 h after infection ( Figure 2B ,C). However, by 72 h, viral replication among TSG101-suppressed and control cells was not significantly different ( Figure 2D ). The return of PRRSV replication in TSG101-suppressed cells might result from virus-stimulated expression of TSG101 and/or incomplete suppression. The earlier and more quantitative comparison of PRRSV replication among TSG101-suppressed and control cells was conducted with the Rluc-expressing PRRSV. Significantly retarded viral replication was detected as early as 5 h post Rluc-PRRSV infection with the in vivo Rluc substrate. The difference in PRRSV replication was quantitatively correlated to the TSG101 levels in different group of cells (Figure 2A and 2E), and could be monitored until 20 h post infection when the substrate was limited. Notably, the replication retardation of indicator protein-expressing viruses in TSG101-silent cells was mostly due to TSG101 suppression because these bioengineered viruses have similar replication kinetics as their parental viruses in normal cells (Figure 1 ). Using the kinetics (i.e., 12-20 h post infection) defined by the indicator protein-expressing viruses, we reproducibly detected retarded replication rates of wildtype PRRS viruses in TSG101 suppressed cells (data no shown). To further test the role of TSG101 in PRRSV infection in porcine cells or pigs, we have characterized the porcine TSG101 cDNA sequence (GenBank TM accession numbers JN882576). The full-length porcine TSG101 cDNA is 1580 bp encoding a precursor protein of 391 residues. TSG101 genes are conserved with most mammalian homologs sharing >94% identity at both RNA and protein levels. The shRNA template sequence we used for loss-of-function studies in MARC-145 cells is identical among human, monkey and porcine TSG101 cDNA. Transient transformation of the shRNA into porcine mDCs similarly suppressed GFP, DsRed-and Rluc-expressing PRRSV until 48 h post infection (data not shown). 