Mutagenesis analysis of the murine leukemia virus matrix protein: identification of regions important for membrane localization and intracellular transport

Abstract

We have created two sets of substitution mutations in the Moloney murine leukemia virus (Mo-MuLV) matrix protein in order to identify domains involved in association with the plasma membrane and in incorporation of the viral envelope glycoproteins into virus particles. The first set of mutations was targeted at putative membrane-associating regions similar to those of the human immunodeficiency virus type 1 matrix protein, which include a polybasic region at the N terminus of the Mo-MuLV matrix protein and two regions predicted to form beta strands. The second set of mutations was created within hydrophobic residues to test for the production of virus particles lacking envelope proteins, with the speculation of an involvement of the membrane-spanning region of the envelope protein in incorporation into virus particles. We have found that mutation of the N-terminal polybasic region redirected virus assembly to the cytoplasm, and we show that tryptophan residues may also play a significant role in the intracellular transport of the matrix protein. In total, 21 mutants of the Mo-MuLV matrix protein were produced, but we did not observe any mutant virus particles lacking the envelope glycoproteins, suggesting that a direct interaction between the Mo-MuLV matrix protein and envelope proteins either may not exist or may occur through multiple redundant interactions.

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

“…The MA protein of type C retroviruses has been suggested to play a critical role in transporting Gag polyproteins to the plasma membrane. Specifically, the basic residues, as well as myristic acid modification within the amino-terminal region of the protein, were found to form a bipartite plasma membranetargeting signal (14,28,38,41,42). In the present study, we found that this signal appears to still be functional even with an amino acid insertion after the basic patch of mutant MuLVs.…”

“…The data described above strongly indicate that an insertion of 18 type D-specific amino acids into type C Gag polyproteins altered the viral morphogenesis to preassemble an immature capsid within the cytoplasm. However, these biochemical approaches could not distinguish between intracytoplasmic capsids that had assembled at a cytosolic site, as in type B and D viruses, and intracisternal capsids that had been released into cytoplasmic vacuoles, as in mutant type C viruses (3,10,12,38). Thus, the effects of these amino acids on capsid assembly were more specifically evaluated by EM studies of COS cells transfected with wild-type or mutant proviral DNAs.…”

“…Our previous studies of MPMV demonstrated that a singleamino-acid substitution (mutant R55W) in the matrix protein (MA) could alter the capsid assembly site of type D viruses from a region within the cytoplasm to the inner surface of the plasma membrane (32). Others have reported that human immunodeficiency virus and Moloney murine leukemia virus (MoMuLV) with amino acid substitutions or deletions in MA undergo capsid assembly within cytoplasmic vacuoles rather than at the plasma membrane (3,10,12,38). These studies strongly suggest that MA can play an essential role in determining the intracellular transport of Gag polyproteins to the site of capsid assembly.…”

A replication-incompetent Rift Valley fever vaccine: Chimeric virus-like particles protect mice and rats against lethal challenge

“…chimVLPs containing a retroviral gag protein (either MoMLV or simian immunodeficiency virus (SIV) gag) and the antigen of interest (e.g., influenza hemagglutinin and neuraminidase) have been recently described (Guo et al, 2003;Haynes et al, 2009). Unfortunately, the generation of RVF chimVLPs is more complicated because RVFV G and MoMLV gag localize to the Golgi (Gerrard and Nichol, 2002;Schmaljohn and Hooper, 2001;Wasmoen et al, 1988) and plasma membranes (Soneoka et al, 1997), respectively, in mammalian cells. However, over-expression of RVFV G leads to some G N /G C localization at the cell surface (Filone et al, 2006;Gerrard and Nichol, 2002;Gerrard and Nichol, 2007;Liu et al, 2008), which allows the generation of RVF chimVLPs.…”

Interaction of Moloney murine leukemia virus matrix protein with IQGAP

“…The Gag proteins of retroviruses, and the matrix protein (MA) domains of Gag in particular, have been long recognized as important for the intracellular trafficking of the viral genome (Yuan et al , 1993). Gag proteins are synthesized in the infected cell as a large precursor, and the N‐terminal MA domain of that precursor directs Gag to the plasma membrane (Spearman et al , 1994; Zhou et al , 1994; Ono and Freed, 1999) using a multipartite membrane‐binding signal: a myristic acid moiety covalently attached to the N‐terminal glycine of MA (Rein et al , 1986), a stretch of hydrophobic residues, and basic residues thought to interact with the acidic phospholipids on the inner leaflet of the plasma membrane (Soneoka et al , 1997). The multimerization of Gag during assembly is thought to promote the higher affinity association with the membrane (Ono et al , 2000).…”