Abstract
The Gag protein of the mouse mammary tumor virus (MMTV) is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV), assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A) resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.
Highlights
The Gag protein plays a pivotal role in dictating the subcellular localization of immature capsid assembly [1,2,3]
Subcellular localization studies of mammary tumor virus (MMTV) Gag have been hampered by insufficient expression levels from Gag-encoding constructs, which is due in part to the lack of cis- and trans-regulatory stabilizing elements that facilitate export of unspliced mRNA from the nucleus to the cytoplasm [37, 38]
While the Gag-GFP protein was undetectable in lysates from cells transfected with Gag-GFP (Fig 1B), the insertion of MMTV Gag 5’ untranslated region (UTR) dramatically increased the accumulation of Gag-GFP to levels similar to those with Mason–Pfizer monkey virus (M-PMV) cytoplasmic transport element (CTE) (Fig 1B and 1C)
Summary
The Gag protein plays a pivotal role in dictating the subcellular localization of immature capsid assembly [1,2,3]. Betaretroviruses assemble immature capsids in the cytoplasm while alpharetroviruses, gammaretroviruses and lentiviruses assemble at the inner plasma membrane. Even though betaretroviruses have a myristoylation signal, the immature capsids assemble intra-cellularly as a result of a cytoplasmic targeting/retention signal (CTRS) in the MA domain [1, 2, 11]. This site was first discovered in the Mason–Pfizer monkey virus (M-PMV) using mutational analyses resulting redistribution of viral assembly from the cytoplasm to the plasma membrane [1, 3]. The Gag polyproteins of Jaagsiekte sheep retrovirus (JSRV) and foamy virus (FV) were found to assemble as capsids at the pericentriolar region [12,13,14,15,16], suggesting that this might be a conserved site for retroviral assembly
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