We formulated an experimental approach in which the fates of multiple viral core components can be tracked in infected cells, with the aim of understanding how TRIM5α restricts retroviral infection. The approach is similar in principle to the “fate of capsid” assay 
, in which the putative separation of viral cores from infected cell lysates on sucrose gradients enables the analysis of their composition. However, our assay is more elaborate, and perhaps more effective, in several aspects. First, we monitored TRIM5α- and TRIMCyp-induced changes not only for CA, but also for IN, viral RNA and reverse transcription products in the same fractionation experiment. Second, in our assay, all of the input cellular material is analyzed, without the need for exclusion of putatively endocytosed virions. Although it is generally held that the majority of retroviral particles become trapped in endosomes of target cells, complicating analysis of early events in infection, this did not seem to be a major problem in our experiments. Indeed, the nearly complete disappearance of IN at T
2 h specifically from restricting cells, argues that there is very little virus associated with the cells that had not reached the cytoplasm by this time point. Although the reasons for this are not clear, possibilities include highly efficient VSV-G-mediated entry in pgsA cells, particular instability of endocytosed virions in pgsA cells, or the fairly low MOIs used in these experiments. Third, infections are fully synchronized and the unbound input virus is removed before infection, which could limit the number of virions that are nonspecifically endocytosed. Fourth, analysis is carried out at an early time (2 h) after infection when events relevant to TRIM5 restriction occur 
. Fifth, we have incorporated quantitative aspects in our experimental system: Q-PCR analysis of viral RNA has proven to be an accurate and quantitative indicator of the fate of the viral core undergoing TRIM5α restriction. Overall our findings suggest that of all of the above components are present in a large complex comprising all or part of the virion core that is a functional intermediate in the infection pathway.
Our findings provide insight into events that take place during TRIM5α restriction (). In parallel with previous findings 
, we observed that N-MLV CA was redistributed from large complexes to soluble fractions in cells expressing huTRIM5α (). We expanded these observations and show that viral RNA was released from large complexes as a result of huTRIM5α restriction (). In contrast, MLV IN was not retained in a soluble form following its loss from dense fractions, and appeared to be degraded (, ).
Model for mechanism of restriction by TRIM5 proteins.
HIV-1 differed from MLV in that neither HIV-1 CA nor viral RNA was apparently increased in soluble fractions concurrent with their loss from large complexes (, ). However, the comparative pre-existing abundance of CA in soluble fractions may have masked any redistribution of CA protein to those fractions. Possible reasons for the discrepant fate of MLV and HIV-1 RNA under restricting conditions are discussed below. In the case of HIV-1 IN, the protein was lost from cells under restricting conditions in much the same way as was observed for MLV. Collectively these results indicate that TRIM5α causes both disassembly and degradation of viral components with similarities and differences in the fates of individual core components across retroviral genera ().
Recent findings have suggested the possibility that the uncoating of retroviral cores early after infection is stimulated by reverse transcription 
and that rhTRIM5α-mediated disassembly of HIV-1 cores requires reverse transcription activity 
. Although in some experiments reverse transcriptase inhibitors modestly increased the amount of capsid detected by western blotting, we did not observe any effect of RT inhibitors on TRIM5-mediated disassembly/degradation of cores in this study. The reasons underlying the discrepancy between our results and the study by Yang et al. 
are not clear. However, one would predict that reverse transcription is not required for restriction by TRIM5, based on the fact that TRIM5 acts rapidly after entry 
, before majority of reverse transcription is completed.
The precise role of proteasomes in TRIM5-mediated restriction has been difficult to unambiguously determine. As previously demonstrated 
, inhibition of proteasomes in restricting cells restored MLV and HIV-1 reverse transcription (, ). Importantly, we found that proteasome inhibition restored a core complex that is biochemically indistinguishable from unrestricted viral cores, and contained CA, IN and viral RNA (, , ). As such, it is unlikely that TRIM5α mediates the complete disassembly of cores without the aid of proteasomes. Nevertheless, it is clear that proteasomes are not required for restriction by TRIM5α, as MG132 treatment of restricting cells does not restore virus infectivity (
and , , ). Recent findings suggest that TRIM21/TRIM5α chimeras have the propensity to form hexameric lattices on the HIV-1 core, and it is possible that this phenomenon, in itself, constitutes the underlying mechanistic basis for restriction 
. The assembly of such a lattice on the core may block the targeting of viral reverse-transcription or pre-integration complexes to the nucleus, because circular viral DNA forms are not generated during restricted HIV-1 infection under conditions of proteasome inhibition 
. However, because HIV-1 and MLV apparently have different underlying mechanisms of entering the nucleus, it is possible that the other mechanisms that sequester viral DNA (e.g. failure to uncoat) may underlie the inability of HIV-1 or MLV to productively infect restrictive cells under conditions of proteasome inhibition.
It is intriguing that some N-MLV and HIV-1 core components, notably viral RNA (and perhaps CA), have somewhat different fates under restrictive conditions (). A possible explanation for this difference is that N-MLV core components are intrinsically more stable and as such, are degraded at a slower rate after TRIM5α-induced disassembly. Alternatively, rhTRIM5α and omkTRIMCyp may either specifically recruit a cofactor that more efficiently degrades the core components or simply disassemble HIV-1 cores at a faster rate. The loss of both N-MLV and HIV-1 IN in dense fractions without any apparent increase in soluble fractions may reflect the previously reported intrinsic instability of these proteins 
We did not detect obvious ubiquitinylation of any core proteins undergoing restriction in our assays. It is conceivable that ubiquitin-independent degradation or disassembly by proteasomes may be important for the observed effects on the cores 
. Alternatively, if TRIM5 is responsible for ubiquitin modification of only a small fraction of core-associated proteins (e.g. CA), we would not be able to detect this modification yet it could be responsible for core disassembly.
The most striking difference between HIV-1 and MLV restriction is the fate of the viral RNA following its release from the core. It appears that MLV RNA is largely preserved, in a soluble form, whereas HIV-1 RNA is lost. We speculate that the mechanism by which HIV-1 viral RNA is lost during restriction is related to its nucleotide composition. It has long been known that the high AU content destabilizes the HIV-1 genome 
. It is therefore conceivable that once the HIV-1 genome is exposed in the cytosol as a result of restriction, AU-rich elements may lead to the degradation of the genome, in the same way as has been observed with several RNAs coding for oncoproteins and growth factors 
. Alternatively, proteasomes themselves, which have been suggested to comprise RNase activity, or other putative TRIM5α associated RNase activities may lead to selective degradation of AU-rich viral RNA molecules 
. Nevertheless, it is unlikely that RNA degradation is critical for TRIM5 restriction as TRIMCypA chimeras containing the RBCC domain from other TRIM proteins, certain RING domain mutants of TRIM5α and squirrel monkey TRIM5α can restrict HIV-1 and SIVmac
infection, respectively, after reverse transcription is completed 
TRIM5α mediated restriction serves as a useful model on which to base investigations of post-entry events. As such, the assay developed here could also be utilized to study restriction-independent events in newly infected cells. For example, it has been suggested that retroviral cores are optimally stable, and changes in CA stability in vitro can lead to defects in reverse transcription 
. The assay developed here could identify the effects of such changes on multiple viral core components in infected cells. However, a caveat of our assay is that the precise nature of the ‘large complexes’ to which we refer is not known. For instance, it is not known whether the large complexes containing CA and the cofractionating core components actually represent intact conical viral cores. Previous investigations of cores isolated from extracellular virions and infected cells revealed notable differences in the density of N-MLV, B-MLV and HIV-1 ‘cores’ 
. We did not observe such differences in our assays, as separation of cytosolic extracts in our experimental system is based on size, rather than density. Therefore, it is plausible that MLV and HIV-1 cores of different densities migrate almost identically on the sucrose gradients as they have similar sizes. Notably, even under non-restrictive conditions, a significant fraction of CA is present in soluble fractions. A similar phenomenon has been previously observed by others during isolation and biochemical characterization of HIV-1, and to a lesser extent MLV, reverse transcription complexes in infected cells 
This could be a consequence of the disassembly of some fraction of CA immediately upon infection or of the fact that only a proportion of the virion CA protein is actually assembled into cores in mature virions 
. It is unlikely that the soluble CA represents complete disintegration of a fraction of viral cores in dense sucrose gradients 
, as neither viral RNA nor IN is solubilized under non-restrictive conditions.
Overall, we devised a novel experimental approach in which events that take place during TRIM5α restriction can be analyzed, and that can be applied generically to the study of early events in retrovirus replication cycle. Our results indicate that viral core components have distinct fates during TRIM5α restriction and are either disassembled or degraded. Importantly, in line with the two-step mechanism previously proposed 
, although the TRIM5α-induced biochemical changes on the viral cores in our assays are sensitive to proteasome inhibition, proteasomal degradation is clearly not required for restriction. Future studies will address by which mechanism TRIM5α can restrict retrovirus infection as well as the mechanistic details of how different core components are affected by restriction.