Membrane Insertion and Transport of Viral Glycoproteins: A Mutational Analysis

The capsids of Mason-Pfizer monkey virus (M-PMV), an immunosuppressive type D retrovirus, are preassembled in the infected cell cytoplasm and are then transported to the plasma membrane, where they are enveloped in a virus glycoprotein-containing lipid bilayer. The role of viral glycoprotein in intracellular transport of M-PMV capsids was investigated with a spontaneous mutant (5A) of M-PMV, which we show here to be defective in envelope glycoprotein biosynthesis. DNA sequence analysis of the env gene of mutant 5A reveals a single nucleotide deletion in the middle of the gene, which results in the synthesis of a truncated form of the envelope glycoprotein. Evidence is presented showing that the mutant glycoprotein is not expressed at the cell surface but is retained in the endoplasmic reticulum. Normal levels of gag-pro-pol precursor polyproteins are made and processed in mutant genome-transfected cells, and high levels of noninfectious particles lacking viral glycoprotein are released with normal kinetics into the culture medium. No intracisternal budding of capsids is observed. We conclude that viral glycoprotein is required neither for targeting preassembled capsids of M-PMV to the plasma membrane for final maturation nor for the budding process. Since the presence or absence of M-PMV glycoprotein at the site of budding does not affect the efficiency or kinetics of the targeting process, the preassembled capsid of M-PMV, in contrast to those of intracisternal type A particles, appears to have an intrinsic signal for intracellular transport to the plasma membrane.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane

Rhee

Hui

Hunter

1990

“…While this model predicts that only 24 (H 146 to Q 170 ) amino acids will span the membrane and act as the membrane anchor, it is supported by our previous observation (9) that a mutant TM with leucine 165 (L 165 ) replaced by an arginine (L165R) remained firmly anchored in the membrane. Moreover, deletions within the MSDs of both the RSV Env and VSV G proteins (1,24) and studies with synthetic anchor domains (9) have demonstrated that 14 to 16 amino acids are sufficient to act as anchor sequences. If this shorter and slightly N-terminally shifted domain does indeed act as a membrane anchor in the molecule, then our previous cytoplasmic trunca- PALMITOYLATION OF THE RSV TM GLYCOPROTEIN 11551 tion of 22 amino acids (42) would leave a residual cytoplasmic domain of 6 amino acids that could continue to interact, directly or indirectly, with capsid proteins during assembly.…”

Section: Discussionmentioning

confidence: 99%

Palmitoylation of the Rous Sarcoma Virus Transmembrane Glycoprotein Is Required for Protein Stability and Virus Infectivity

Ochsenbauer-Jambor

Miller

Roberts

et al. 2001

Self Cite

The Rous sarcoma virus (RSV) transmembrane (TM) glycoprotein is modified by the addition of palmitic acid. To identify whether conserved cysteines within the hydrophobic anchor region are the site(s) of palmitoylation, and to determine the role of acylation in glycoprotein function, cysteines at residues 164 and 167 of the TM protein were mutated to glycine (C164G, C167G, and C164G/C167G). In CV-1 cells, palmitate was added to env gene products containing single mutations but was absent in the double-mutant Env. Although mutant Pr95 Env precursors were synthesized with wild-type kinetics, the phenotypes of the mutants differed markedly. Env-C164G had properties similar to those of the wild type, while Env-C167G was degraded faster, and Env containing the double mutant C164G/C167G was very rapidly degraded. Degradation occurred after transient plasma membrane expression. The decrease in steady-state surface expression and increased rate of internalization into endosomes and lysosomes paralleled the decrease in palmitoylation observed for the mutants. The phenotypes of mutant viruses were assessed in avian cells in the context of the pATV8R proviral genome. Virus containing the C164G mutation replicated with wild-type kinetics but exhibited reduced peak reverse transcriptase levels. In contrast, viruses containing either the C167G or the C164G/C167G mutation were poorly infectious or noninfectious, respectively. These phenotypes correlated with different degrees of glycoprotein incorporation into virions. Infectious revertants of the double mutant demonstrated the importance of cysteine-167 for efficient plasma membrane expression and Env incorporation. The observation that both cysteines within the membrane-spanning domain are accessible for acylation has implications for the topology of this region, and a model is proposed.

“…Sequences immediately upstream and downstream of the TM domain could be involved in controlling those changes and/or interaction. It has been shown that mutations of certain amino acids, particularly the positively charged residues, in the vicinity of the TM domain of a TM molecule could drastically affect its membrane-anchoring stability and even revert its membrane orientation (19,33). Deletion of the sequences immediately upstream of the TM domains of the CCros and CVros proteins may release the physical constraint of these TM molecules and render them constitutively active in kinase activity.…”

Section: Discussionmentioning

confidence: 99%

“…Protein analysis. Metabolic labeling, protein extraction, subcellular fractionation, immunoprecipitation, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and in vitro kinase assays were done according to published procedures (11,(13)(14)(15)(16)(17)(18)(19)(20)22). For glycosylation-inhibiting experiments, cells were pretreated with tunicamycin (10 jLg/ml; Sigma) for 2 h and then [35S]methionine labeled for 4 h in the presence of tunicamycin.…”

Section: Methodsmentioning

confidence: 99%

Molecular and biochemical bases for activation of the transforming potential of the proto-oncogene c-ros

Zong

Poon

Chen

et al. 1993

The transforming gene of avian sarcoma virus UR2, v-ros, encodes a receptor-like protein tyrosine kinase and differs from its proto-oncogene, c-ros, in its 5' truncation and fusion to viral gag, a three-amino-acid (aa) insertion in the transmembrane (TM) domain, and changes in the carboxyl region. To explore the basis for activation of the c-ros transforming potential, various c-ros retroviral vectors containing those changes were constructed and studied for their biological and biochemical properties. Ufcros codes for the full-length c-ros protein of 2,311 aa, Uppcros has 1,661-aa internal deletion in the extracellular domain, CCros contains the 3' c-ros cDNA fused 150 aa upstream of the TM domain to the UR2 gag, CVros is the same as CCros except that the 3' region is replaced by that of v-ros, and VCros is the same as CCros except that the 5' region is replaced by that of v-ros. The Ufcros, Uppcros, CCros, and CVros are inactive in transforming chicken embryo fibroblasts, whereas VCros is as potent as UR2 in cell-transforming and tumorigenic activities. Upon passages of CCros and CVros viruses, the additional extracellular sequence in comparison with that of v-ros was deleted; concurrently, both viruses (named CC5d and CV5d, respectively) attained moderate transforming activity, albeit significantly lower than that of UR2 or VCros. The native c-ros protein has a very low protein tyrosine kinase activity, whereas the ppcros protein is constitutively activated in kinase activity. The inability of CCros and CVros to transform chicken embryo fibroblasts is consistent with the inefficient membrane association, instability, and low kinase activity of their encoded proteins. The CC5d and CV5d proteins are indistinguishable in kinase activity, membrane association, and stability from the v-ros protein. The reduced transforming potency of CC5d and CV5d proteins can be attributed only to their differential substrate interaction, notably the failure to phosphorylate a 88-kDa protein. We conclude that the 5' rather than the 3' modification of c-ros is essential for its oncogenic activation; the sequence upstream of the TM domain has a negative effect on the transforming activity of CCros and CVros and needs to be deleted to activate their biological activity. The proto-oncogene c-ros is the cellular counterpart of the oncogene v-ros of avian sarcoma virus (ASV) UR2 (30, 31). Sequence analysis of the chicken and human genomic DNA clones representing the 3' portion of c-ros revealed that it is a receptor-like protein tyrosine kinase (PTK) (28, 30). Recently, the c-ros cDNAs from rat (29), human (3), and chicken (5) have been isolated and sequenced. Those results confirm the initial suggestion that c-ros codes for a receptor-like PTK. However, it differs from other well-characterized receptor PTKs (RPTKs) such as the insulin receptor (IR) and insulinlike growth factor I receptor (IGFR) (8, 40, 42), epidermal growth factor receptor (41), platelet-derived growth factor receptor (47), and colony-stimulating factor receptor (6, 1...