Kakoli Ghosh

The Membrane-Proximal Region of Vesicular Stomatitis Virus Glycoprotein G Ectodomain Is Critical for Fusion and Virus Infectivity

ABSTRACT The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.

Role of modified nucleoside adjacent to 3t́-end of anticodon in codon-anticodon interaction

Abstract Removal of the base Y adjacent to the anticodon of yeast tRNAPhe changes its coding properties. Modified tRNAPhe responds to the codon UUC better than the codon UUU. The codon UUU can be read efficiently only at higher Mg2+ conc. or in the presence of streptomycin. Binding studies show that the unmodified and the modified tRNAPhe can not occur next to each other on the two binding sites of a ribosome. Removal of Y changes the configuration of the anticodon loop of tRNAPhe such that the base (G) involved in “Wobble” changes its relative orientation to the other bases in the anticodon.

Membrane fusion activity of vesicular stomatitis virus glycoprotein G is induced by low pH but not by heat or denaturant.

The fusogenic envelope glycoprotein G of the rhabdovirus vesicular stomatitis virus (VSV) induces membrane fusion at acidic pH. At acidic pH the G protein undergoes a major structural reorganization leading to the fusogenic conformation. However, unlike other viral fusion proteins, the low-pH-induced conformational change of VSV G is completely reversible. As well, the presence of an alpha-helical coiled-coil motif required for fusion by a number of viral and cellular fusion proteins was not predicted in VSV G protein by using a number of algorithms. Results of pH dependence of the thermal stability of G protein as determined by intrinsic Trp fluorescence and circular dichroism (CD) spectroscopy show that the G protein is equally stable at neutral or acidic pH. Destabilization of G structure at neutral pH with either heat or urea did not induce membrane fusion or conformational change(s) leading to membrane fusion. Taken together, these data suggest that the mechanism of VSV G-induced fusion is distinct from the fusion mechanism of fusion proteins that involve a coiled-coil motif.

Initiation of protein synthesis in eukaryotes

Abstract The two major species of tRNAMet from wheat germ have been purified and studied for their role in protein synthesis. tRNA1Met can be charged both by wheat embryo synthetase and E. coli synthetase and tRNA2Met can be charged only by wheat embryo synthetase. Methionyl-tTNA1Met fails to transfer methionine into a polypeptide chain while met-tRNA2Met can transfer methionine into a polypeptide chain. At low Mg+2 concentration the transfer of methionine from met-tRNA2Met is dependent on the incorporation of methionine from met-tRNA1Met at the N-terminal position.

Mutations in the conserved carboxy-terminal hydrophobic region of glycoprotein gB affect infectivity of herpes simplex virus.

Glycoprotein gB is the most highly conserved glycoprotein in the herpesvirus family and plays a critical role in virus entry and fusion. Glycoprotein gB of herpes simplex virus type 1 contains a hydrophobic stretch of 69 aa near the carboxy terminus that is essential for its biological activity. To determine the role(s) of specific amino acids in the carboxy-terminal hydrophobic region, a number of amino acids were mutagenized that are highly conserved in this region within the gB homologues of the family HERPESVIRIDAE: Three conserved residues in the membrane anchor domain, namely A786, A790 and A791, as well as amino acids G743, G746, G766, G770 and P774, that are non-variant in Herpesviridae, were mutagenized. The ability of the mutant proteins to rescue the infectivity of the gB-null virus, K082, in trans was measured by a complementation assay. All of the mutant proteins formed dimers and were incorporated in virion particles produced in the complementation assay. Mutants G746N, G766N, F770S and P774L showed negligible complementation of K082, whereas mutant G743R showed a reduced activity. Virion particles containing these four mutant glycoproteins also showed a markedly reduced rate of entry compared to the wild-type. The results suggest that non-variant residues in the carboxy-terminal hydrophobic region of the gB protein may be important in virus infectivity.

Specificity of the initiator methionine tRNA for terminal and internal recognition.

Initiator methionyl-tRNAs from E.,coli and wheat embryo can also transfer methionine into internal positions of a polypeptide chain. This transfer depends on the protein synthesizing system used, an E.,coli system being about 10-times more active than a wheat embryo system. At low Mg2+ concentration this incorporation depends on the presence of both initiation factors and fmet-tRNAfMet, but the rate is slower than that from met-tRNAmMet. Wheat embryo initiator met-tRNA1Met can initiate protein synthesis in an E.,coli system only when it is chemically formylated, but fmet-tRNA2Met (non-initiator) is inactive in this reaction.