Infectious entry of enveloped viruses is thought to proceed by one of two mechanisms. pH-dependent viruses enter the cells by receptor-mediated endocytosis and are inhibited by transient treatment with agents that prevent acidification of vesicles in the endocytic pathway, while pH-independent viruses are not inhibited by such agents and are thought to enter the cell by direct fusion with the plasma membrane. Nearly all retroviruses, including amphotropic murine leukemia virus (MuLV) and human immunodeficiency virus type 1, are classified as pH independent. However, ecotropic MuLV is considered to be a pH-dependent virus. We have examined the infectious entry of ecotropic and amphotropic MuLVs and found that they were equally inhibited by NH 4 Cl and bafilomycin A. These agents inhibited both viruses only partially over the course of the experiments. Agents that block the acidification of endocytic vesicles also arrest vesicular trafficking. Thus, partial inhibition of the MuLVs could be the result of virus inactivation during arrest in this pathway. In support of this contention, we found that that the loss of infectivity of the MuLVs during treatment of target cells with the drugs closely corresponded to the loss of activity due to spontaneous inactivation at 37°C in the same period of time. Furthermore, the drugs had no effect on the efficiency of infection under conditions in which the duration of infection was held to a very short period to minimize the effects of spontaneous inactivation. These results indicate that the infectious processes of both ecotropic and amphotropic MuLVs were arrested rather than aborted by transient treatment of the cells with the drugs. We also found that infectious viruses were efficiently internalized during treatment. This indicated that the arrest occurred in an intracellular compartment and that the infectious process of both the amphotropic and ecotropic MuLVs very likely involved endocytosis. An important aspect of this study pertains to the interpretation of experiments in which agents that block endocytic acidification inhibit infectivity. As we have found with the MuLVs, inhibition of infectivity may be secondary to the block of endocytic acidification. While this strongly suggests the involvement of an endocytic pathway, it does not necessarily indicate a requirement for an acidic compartment during the infectious process. Likewise, a lack of inhibition during transient treatment with the drugs would not preclude an endocytic pathway for viruses that are stable during the course of the treatment.
The cytoplasmic tail of the immature Moloney murine leukemia virus (MoMuLV) envelope protein is approximately 32 amino acids long. During viral maturation, the viral protease cleaves this tail to release a 16-amino-acid R peptide, thereby rendering the envelope protein fusion competent. A series of truncations, deletions, and amino acid substitutions were constructed in this cytoplasmic tail to examine its role in fusion and viral transduction. Sequential truncation of the cytoplasmic tail revealed that removal of as few as 11 amino acids resulted in significant fusion when the envelope protein was expressed in NIH 3T3 cells, similar to that seen following expression of an R-less envelope (truncation of 16 amino acids). Further truncation of the cytoplasmic tail beyond the R-peptide cleavage site toward the membrane-spanning region had no additional effect on the level of fusion observed. In contrast, some deletions and nonconservative amino acid substitutions in the membrane-proximal region of the cytoplasmic tail (residues L602 to F605) reduced the amount of fusion observed in XC cell cocultivation assays, suggesting that this region influences the fusogenicity of full-length envelope protein. Expression of the mutant envelope proteins in a retroviral vector system revealed that decreased envelope-mediated cell-cell fusion correlated with a decrease in infectivity of the resulting virions. Additionally, some mutant envelope proteins which were capable of mediating cell-cell fusion were not efficiently incorporated into retroviral particles, resulting in defective virions. The cytoplasmic tail of MoMuLV envelope protein therefore influences both the fusogenicity of the envelope protein and its incorporation into virions.
The mouse TSH beta gene contains two start sites of transcription and exhibits alternative splicing among its first three exons, which encode 5'-untranslated mRNA sequences. Expression of the mouse TSH beta gene, therefore, gives rise to multiple mRNAs, each with a unique 5'-untranslated region. We have determined the relative translational efficiencies of these mRNAs in vitro, and we demonstrate that one of them directs the synthesis of a novel TSH beta presubunit. The four TSH beta mRNAs that are expressed from the down-stream transcription start site (TSS2) and the major mRNA derived from the up-stream start site (TSS1) were transcribed in vitro and translated in reticulocyte lysates and wheat germ extracts. The mRNA from TSS1 gave a novel TSH beta presubunit due to initiation of translation at an up-stream AUG unique to this mRNA. The novel presubunit contained a 17-amino acid NH2-terminal extension sequence, compared to the normal TSH beta presubunit, which is encoded by each of the mRNAs from TSS2. Despite the fact that the NH2-terminal extension sequence appeared to lack the characteristics of a signal peptide, the novel TSH beta presubunit was processed about 50% as efficiently by microsomal membranes as the normal presubunit, with glycosylation and cleavage by signal peptidase. There was an approximately 2-fold range in relative translatability among the different TSH beta mRNAs, and the mRNA encoding the novel TSH beta presubunit had the highest translational efficiency. Our data, therefore, suggest that the longer presubunit may be synthesized in vivo in significant amounts and give rise to a novel mature TSH beta subunit.
Expression of the single mouse TSH beta gene gives rise to multiple mRNAs, and we have previously shown that in vitro, one of these mRNAs gives rise to a novel TSH beta-presubunit due to initiation of translation at an in-frame start site unique to this mRNA which is up-stream of the normal start site. The novel presubunit contains a 17-amino acid NH2-terminal extension sequence compared to the normal presubunit. Although this extension sequence does not have the characteristics of a normal signal sequence, the novel TSH beta-presubunit was processed in vitro by microsomal membranes. In this study we have examined the translation product of this mRNA in intact cells and whether in vivo it gives rise to a processed secreted TSH beta-subunit that has an NH2-terminal sequence different from that of the established TSH beta-subunit. Firstly, mRNAs encoding alpha-presubunit and either the normal or novel TSH beta-presubunit were microinjected into Xenopus oocytes, and it was found that immunoprecipitable TSH dimer was secreted into the medium regardless of the mRNA used for TSH beta-subunit synthesis. However, less TSH was obtained when the TSH beta-subunit was derived from the extended TSH beta-presubunit. Secondly, when COS cells were transiently transfected with plasmids expressing alpha-presubunit and either the normal or novel TSH beta-presubunit, secreted TSH was obtained when the TSH beta-subunit was derived from either presubunit. TSH dimer was also obtained when the TSH beta-presubunit was derived from a mRNA encoding the extended presubunit in which the down-stream AUG had been eliminated by site-specific mutagenesis. This demonstrated that the up-stream translation start site was used in the intact cell and that secreted TSH beta-subunit was derived from the extended presubunit and not from normal presubunit resulting from translational readthrough to the down-stream AUG. When secreted TSH beta-subunits derived from the normal and extended TSH beta-presubunits were digested with endoproteinase LysC, the NH2-terminal fragments were similar in size, suggesting that the NH2-terminal extension had little if any effect on the site of cleavage by signal peptidase. Our data, therefore, demonstrate that the longer TSH beta-presubunit is synthesized in vivo and strongly suggest that it is processed in the intact cell to give a mature secreted TSH beta-subunit indistinguishable from that derived from the normal TSH beta-presubunit.
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