Substitution of leucine 8 in the simian immunodeficiency virus matrix protein impairs particle formation without affecting N-myristylation of the Gag precursor.
In the late stages of replication of the simian immunodeficiency viruses (SIV), the matrix protein (MA) plays a central role in the transport of Pr55gag to the plasma membrane, assembly of virus particles, and incorporation of the envelope glycoprotein into particles. Targeting of Pr55gag to the plasma membrane is mediated by two motifs within the MA protein: the N-terminal myristate and a cluster of positively charged amino acids. In this report, we characterized the assembly phenotype of an SIV Gag mutant (L8Q) carrying the single amino acid substitution of glutamine for leucine at position 8 in the MA domain. The hydropathic profile of the mutated MA protein indicated that the L8Q amino acid change disrupts the hydrophobic character of the region comprising the first 10 residues of the protein. Expression of mutant L8Q Gag protein in CV-1 cells, by means of the vaccinia virus vector system, resulted in efficient synthesis and N-myristylation of Pr55gag. However, this mutation severely impaired particle production, as inferred from both biochemical and electron microscopy analyses. Cellular fractionation assays revealed that in cells expressing mutant L8Q, the proportion of cytosol-associated Pr55gag was significantly increased compared to that observed upon expression of wild-type Gag. Furthermore, mutant L8Q Gag partitioned onto cytosol and membrane fractions in a manner similar to nonmyristylated Gag polyprotein. Taken together, these results indicate that the L8Q mutation reduces the membrane-binding capacity of the Gag precursor. It is therefore likely that in the SIV MA, in addition to the N-myristate group, the hydrophobicity of the neighboring region is important for efficient association of Pr55gag with the plasma membrane.
González
S A
SA
Centro de Virología Animal (CEVAN-CONICET), Buenos Aires, Argentina. sag@cevan.sld.ar
Affranchino
J L
JL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D015683:Gene Products, gag / Q000378:metabolism*; D007668:Kidney; D007930:Leucine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015302:Simian Immunodeficiency Virus / Q000502:physiology* / Q000648:ultrastructure; D014162:Transfection; D014616:Vaccinia virus; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication
D015683:Gene Products, gag; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D019814:Myristic Acid; D007930:Leucine