Recent progress in cryo-TEM, cryo-ET and 3 D reconstruction has led to many major breakthroughs in our understanding of virus structure. For instance, the architecture of immature and mature HIV-1 [13
], murine leukemia virus (MuLV) [25
], and Rous sarcoma virus (RSV) [26
] has been investigated in great detail. Although HTLV-1 was the first human retrovirus to be discovered [27
], very little is known about HTLV-1 virion morphology. Progress in this area of HTLV-1 biology has been hampered due to the fragile nature of HTLV-1 proviral sequences as well as limited levels of viral gene expression and viral replication in tissue culture. HTLV-1 pathogenesis is typically observed decades after infection with low viral loads. In fact, studies have shown that HTLV-1 restricts its own gene expression via viral regulatory factors [29
]. HTLV-1 has likely evolved such a replication strategy for immune escape. Furthermore, high AU-content of the retroviral genome may lead to instability during nuclear transport of mRNAs [31
], which also contributes to the overall low level of viral gene and protein expression. In this study, we have designed a model system to study HTLV-1 Gag trafficking in cells and VLP production and morphology. The basis for this model system is a codon-optimized HTLV-1 Gag-EYFP construct, which can be readily amplified as a plasmid, expresses high levels of HTLV-1 Gag in mammalian cells, and robustly produces VLPs. This is the first model system developed for HTLV-1 for the study of virus particle assembly, release, as well as virus particle morphology.
While our model system does not express Gag in the context of a proviral sequence (i.e., codon-optimized and EYFP-tagged), our results indicate that the VLPs produced have the morphology of the authentic HTLV-1 immature particles. Furthermore, while the Gag trafficking pathways used by the HTLV-1 Gag in this model system may be different from that of Gag expressed from the provirus, the production of VLPs argues that the trafficking pathways are biological relevant since VLP production is the result of expression of the codon-optimized Gag-EYFP fusion. The altered Gag trafficking pathways could influence envelope incorporation into VLPs, though our cryo-TEM data revealed an abundance of VLPs with VSVG. The VLPs characterized in our study resemble immature HTLV-1 and can be readily observed in ultrathin sections of 293Ts transfected with pEYFP-N3 HTLV-1 Gag (Figure ). In addition, cell culture supernatants from 293Ts transiently transfected with pEYFP-N3 HTLV-1 Gag contain high levels of Gag-EYFP fusion proteins (Figure ), which provides second line of evidence for the production of VLPs. In the fraction of sucrose gradients containing the highly-fluorescent material, cryo-TEM reveals that these fractions are highly concentrated with VLPs (Figure ). Expression of EYFP alone in cells did not lead to the release of fluorescence in the cell culture supernatant (data not shown), arguing that we were specifically detecting the Gag-EYFP fusion in the VLPs.
We found that the intracellular localization of HTLV-1 Gag-EYFP was comparable to that of authentic Gag in HeLa cells (Figure ). This implies, though does not formally prove, that there are similarities in the Gag trafficking pathway used by Gag-EYFP and authentic Gag. Among retroviruses, intracellular Gag polyproteins are thought to target and accumulate at membrane compartments prior to viral assembly. In the case of HIV-1, Gag is thought to primarily target specific domains of the plasma membrane where PI(4,5)P2 and cholesterol are enriched, though endosomal trafficking may also play a role. For HTLV-1, several studies have suggested the association of Gag with several markers found on the membranes of late endosomes and multivesicular bodies - these markers are also enriched at the plasma membrane [32
Our cryo-TEM and FFS analysis determined that the average VLP diameter was 71 ± 20 nm and 75 ± 4 nm, respectively. As observed in other retroviruses, the size of HTLV-1-like particles varies greatly, ranging from 30 to 237 nm. According to the size distribution (Figure ), over 25% of the population is between 70-80 nm in diameter, indicating that HTLV-1 is smaller, on average, than previously believed. The average diameter of HTLV-1 has been shown to be anywhere from 95.1 ± 19.0 nm to 110.0 ± 15.5 nm depending on different types of staining used for TEM [19
]. However, morphological details are lost with staining methods when the biological specimens are completely dehydrated. Examining frozen, hydrated samples via cryo-TEM reflects the native morphology of the viral particles. Moreover, FFS offers a unique way to determine the average hydrodynamic radius in the cell supernatant without any special treatment or preparation prior to FFS analysis. The use of two independent methods for determining VLP diameter provides a strong argument in favor of the relatively small particle diameter for the HTLV-1-like particles analyzed in our study.
We used FFS to also investigate Gag stoichiometry in the VLPs by performing brightness analysis of the FFS data. We determined that the average Gag copy number per VLP is ~510, which implies that only half of the available membrane surface is covered by Gag. Brightness analysis further revealed heterogeneity of the Gag copy number by identifying two brightness species. The presence of heterogeneity in the Gag copy number has also been observed for HIV-1 Gag-based VLPs [21
]. Since FFS analysis involves an ensemble average over all measured VLPs, the information in the PCH curve only provides a rough approximation of the true Gag copy number distribution for the VLPs. Thus, the two brightness species identified by PCH analysis do not necessarily reflect two distinct populations of VLPs, but more likely reflect the analytical approximation of a broad distribution of Gag stoichiometries that approximately range from 300 to 880. PCH analysis also demonstrates that only ~20% of VLPs have high copy numbers.
Among the thousands of cryo-TEM images of VLPs examined in our study, we commonly observed particles that did not have electron density consistent with a Gag shell covering the entire membrane surface (Figure ). These results suggest that the majority of HTLV-1 particles analyzed contain an incomplete shell of Gag lattice. In the case of HIV-1, previous 3 D structural analyses revealed that most immature virions contain a continuous, but incomplete, hexameric arranged Gag shell, covering approximately 40-60% of the membrane surface [14
]. The average copy number of Gag per particle was calculated to be approximately 2,400 ± 700 per immature particle. The Gag number increased significantly, however, when defects were introduced during budding [36
]. In fact, the data is in agreement with our previous FFS study indicating that HIV-1 Gag stoichiometry ranges from 750 to 2,500 [21
]. Since the mature core consists of only 1,000-1,500 molecules of CA [23
], it is reasonable to believe that an equivalent number of Gag molecules are needed to form an immature particle. Our current study is the first to provide insights into the structural details for HTLV-1.
studies suggest that the HTLV-1 Gag shell is very likely to be organized differently compared to that of HIV-1 Gag [16
]. When examining the cryo-TEM images of HTLV-1-like particles, we rarely observed a highly ordered Gag lattice next to the lipid bilayer (Figure ), a feature frequently observed in immature HIV-1 particles. The HTLV-1 particles analyzed in our study were fairly uniform in their overall inner density. Furthermore, in contrast to HIV-1, no defined peaks representing the CA or NC domains were found in the HTLV-1 radial density profile. The two peaks representing the lipid bilayers could be clearly determined (Figure ), whereas the inner density profile appeared to be relatively flat. Since cryo-TEM images represent a two-dimensional projection of the virus particle, a more rigorous structural analysis, such as cryo-ET, is needed to further examine the protein organization in the HTLV-1-like particles.
In summary, we have developed the first efficient and robust model system for the analysis of HTLV-1 Gag cellular trafficking, virus particle assembly, release and particle morphology. This system will allow for significant advancements in understanding of the basic mechanisms of HTLV-1 replication - which has been severely hampered due to the limitations in studying HTLV-1 in tissue culture. Our study also represents the first description of immature HTLV-1 particles as well as quantitative measurements of particle size, Gag copy number, and an initial analysis of the HTLV-1 Gag lattice. Future application of cryo-electron tomography will aid in gaining greater insight into HTLV-1 particle morphology. A deeper understanding of the basic mechanisms involved in HTLV-1 particle assembly and morphology should help to enhance our global understanding of the basis of HTLV-1 particle infectivity, transmission and pathogenesis.