Identification of a Cytoplasmic Targeting/Retention Signal in a Retroviral Gag Polyprotein

Abstract

Retroviral capsid assembly can occur by either of two distinct morphogenic processes: in type C viruses, the capsid assembles and buds at the plasma membrane, while in type B and D viruses, the capsid assembles within the cytoplasm and is then transported to the plasma membrane for budding. We have previously reported that a single-amino-acid substitution of a tryptophan for an arginine in the matrix protein (MA) of Mason-Pfizer monkey virus (MPMV) converts its capsid assembly from that of a type D retrovirus to that of the type C viruses (S. S. Rhee and E. Hunter, Cell 63:77–86, 1990). Here we identify a region of 18 amino acids within the MA of MPMV that is responsible for type D-specific morphogenesis. Insertion of these 18 amino acids into the MA of type C Moloney murine leukemia virus causes it to assemble an immature capsid in the cytoplasm. Furthermore, fusion of the MPMV MA to the green fluorescent protein resulted in altered intracellular targeting and a punctate accumulation of the fusion protein in the cytoplasm. These 18 amino acids, which are necessary and sufficient to target retroviral Gag polyproteins to defined sites in the cytoplasm, appear to define a novel mammalian cytoplasmic targeting/retention signal.

The M‐PMV Cytoplasmic Targeting‐Retention Signal Directs Nascent Gag Polypeptides to a Pericentriolar Region of the Cell

“…The cytoplasmic assembly of M‐PMV Gag is mediated by an 18‐amino acid region in the N‐terminus of Gag that we have previously termed the cytoplasmic targeting/retention signal (CTRS) (15). Mutation of an arginine residue to a tryptophan within this element (R55W) alters the morphogenesis of M‐PMV so that capsid assembly now occurs on the plasma membrane (14).…”

“…The cytoplasmic targeting/retention signal or CTRS sequence of the M‐PMV Gag polyprotein was previously demonstrated to be responsible for the intracytoplasmic assembly of retroviruses exhibiting B/D‐type morphogenesis (15,27). Here we show that the CTRS is responsible for dynein‐mediated targeting/retention of nascent Gag polyproteins at the centriolar region of the cell, and that assembly of immature M‐PMV capsids occurs at this location.…”

“…This allows the bipartite targeting signal, contained in the matrix domain of both C‐type and B/D‐type retroviruses, to target Gag to the plasma membrane. Addition of the M‐PMV CTRS, or a homologous CTRS‐like sequence found in mouse mammary tumor virus (MMTV), to a normally C‐type retrovirus (MuLV) results in retargeting of its assembly to the cytoplasm (15). Additionally, a fusion protein consisting of the M‐PMV MA domain and green fluorescent protein (GFP) follows the same pattern as M‐PMV Gag when the corresponding wild‐type or mutant CTRS sequence is present (15).…”

“…Despite the presence of the bipartite signal found in C‐type virus Gag polyproteins, an 18‐amino acid sequence within the MA domain, termed the cytoplasmic targeting/retention signal (CTRS), appears to function as a dominant signal that is responsible for intracytoplasmic assembly of M‐PMV immature capsids. This sequence is conserved within the MA proteins of other B‐ and D‐type Gag polyproteins (14) and insertion of this sequence into the same region of murine leukemia virus (MuLV), which typically exhibits C‐type morphogenesis, results in intracytoplasmic assembly of MuLV Gag (15). Moreover, while C‐type and D‐type morphogenic pathways appear distinct, mutation of a single arginine within the CTRS signal of M‐PMV Gag to a tryptophan residue (R55W), abrogates its dominant targeting mechanism, and M‐PMV Gag follows a C‐type assembly pathway (14).…”

Separate Assembly and Transport Domains within the Gag Precursor of Mason-Pfizer Monkey Virus

“…Further evidence for the transport function of the R55 loop region of M-PMV matrix has been provided by Choi et al 7, who have engineered this region into the matrix domain of murine leukemia virus (MuLV) Gag resulting in the production of intracytoplasmic particles. Similarly, the homologous region of MMTV can also induce the assembly of intracytoplasmic immature particles when engineered into the MuLV matrix domain (7). These results led the authors of that study to term this region a cytoplasmic transport-retention signal (CTRS).…”