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Viral subversion of protein trafficking pathways
News & Comment
TRENDS in Biochemical Sciences Vol.27 No.7 July 2002
335
Journal Club
Viral subversion of protein trafficking pathways Most of us have heard of Simian Virus 40 (SV40), a non-enveloped DNA virus that is best known for its capacity to transform tissue culture cells. SV40 enters mammalian cells by an unusual route: it binds to MHC class I molecules at the cell surface, and is subsequently taken up by non-clathrincoated vesicles by a mechanism that is dependent on caveolin and cholesterol. Early electron microscopy studies show the endocytosed virus accumulating in the endoplasmic reticulum (ER). At the time, this observation was confusing because the virus ultimately needs to enter the nucleus where uncoating and replication occur, and it was unclear how it would reach this destination from the ER lumen; this raised significant doubt as to whether the viral population in the ER was in any way connected to productive infection of the cell. In addition, there was no precedent for a connection between the ER and the endocytic pathway. We now know, however, that a few plant and bacterial toxins, such as cholera toxin, enter cells by endocytosis and travel retrogradely through the secretory pathway all the way to the ER; here, the toxins
camouflage themselves as misfolded proteins and exit through the proteinconducting channel in the ER membrane into the cytosol, where they inactivate their target molecules and thus kill the cell. Can a virus do the same thing? Richards et al. [1] suggest that this could be the case. The authors have investigated the effects of incubation at 20°C and agents interfering with trafficking mediated by coatomer (COP) I- and II-coated vesicles on SV40 infection and cholera toxin toxicity, and in all cases, both processes are equally affected. This suggests that cholera toxin and SV40 do indeed use the same route to the ER, and that this route includes early endosomes, the trans-Golgi network and the Golgi complex itself. Intriguingly, the authors found that a modified dipeptide, which blocks cholera toxin poisoning at a post-Golgi step, also prevents SV40 infection. Incubation with the peptide depletes intracellular Ca2+ stores, and it has been suggested that it directly affect the channels that mediate cholera toxin transport from the ER to the cytosol. So how does a virus resemble a toxin or a misfolded protein? And if it really uses this
exit from the ER, how does SV40 with a diameter of 45 nm make it through the protein-translocation channel which, even by the most generous estimate, has a maximal opening of 8 nm? There is some evidence that at the plasma membrane SV40 can ‘recruit’ caveolin to surround the viral particles before entry. Perhaps it can do the same with protein-translocation channel subunits and generate a giant pore in the ER membrane. The subdomains of the ER in which the virus accumulates are continuous with the rough ER, but not studded with ribosomes, which primarily bind to channels engaged in protein translocation. So either this region of the ER is devoid of protein-translocation channels or the channels in this region have all become SV40 exits. Time and more experimentation will tell. 1 Richards, A.A. et al. (2002) Inhibitors of COP-mediated transport and cholera toxin action inhibit Simian Virus 40 infection. Mol. Biol. Cell 13, 1750–1764
Karin Römisch [email protected]
mRNA decay: the big picture The cell can precisely regulate expression of any given gene by controlling the rates of transcription and mRNA decay. Although mRNA decay rates can differ by a factor of >100, relatively little is known about the global regulation of this process. Now, Wang et al. [1] have started to redress this imbalance by undertaking a global profiling of mRNA turnover in yeast. They found that mRNA decay rates did not generally correlate with any obvious molecular characteristic but instead seemed to relate to the physiological function of the corresponding gene product. The method adopted by Wang et al. was to combine a well-characterized global transcriptional shut-off assay with DNA microarray analyses. A yeast strain containing a temperature-sensitive allele encoding RNA polymerase II (RNA Pol II) was first grown at 24 °C and then shifted to a restrictive temperature for RNA Pol II and http://tibs.trends.com
samples taken at intervals. The lack of RNA Pol II activity at the elevated temperature meant that changes in steady-state mRNA levels would be mainly determined by specific rates of mRNA decay. RNA was extracted from these samples and microarray analysis used to quantitate the levels of 4687 individual mRNAs at each time point and these data used to calculate half-lives for individual species. A parallel series of experiments provided support for a general mechanism of mRNA decay preceded by shortening of poly(A) tails. The rates observed by Wang et al. varied by a factor of >30, confirming the importance of decay in controlling mRNA turnover during gene expression. However, no relationship could be found between these rates and various factors that had previously been suggested to contribute to decay, such as abundance, ORF size or codon usage. Instead, similarities were
found in decay rates for mRNAs encoding proteins within stoichiometric complexes. For example, 13 of the mRNAs corresponding to the 14 proteins of the 20S proteasome have closely matched decay rates and in the most striking example, 126 of the 131 ribosomal protein-encoding mRNAs analysed have closely clustered decay rates (tellingly, four of the five exceptions are unusual in not being targets of the transcription factor Rap1). The robustness of the correlation between decay rates was supported by a more detailed statistical analysis of 95 protein complexes. Beyond these tightly clustered decay rates associated with complexes, the authors also found broad correlations for mRNAs encoding proteins within functional groups. For example, mRNAs encoding metabolic enzymes were generally long-lived, whereas those associated with transient processes such as mating turned over rapidly.
0968-0004/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
TRENDS in Biochemical Sciences Vol.27 No.7 July 2002
335
Journal Club
Viral subversion of protein trafficking pathways Most of us have heard of Simian Virus 40 (SV40), a non-enveloped DNA virus that is best known for its capacity to transform tissue culture cells. SV40 enters mammalian cells by an unusual route: it binds to MHC class I molecules at the cell surface, and is subsequently taken up by non-clathrincoated vesicles by a mechanism that is dependent on caveolin and cholesterol. Early electron microscopy studies show the endocytosed virus accumulating in the endoplasmic reticulum (ER). At the time, this observation was confusing because the virus ultimately needs to enter the nucleus where uncoating and replication occur, and it was unclear how it would reach this destination from the ER lumen; this raised significant doubt as to whether the viral population in the ER was in any way connected to productive infection of the cell. In addition, there was no precedent for a connection between the ER and the endocytic pathway. We now know, however, that a few plant and bacterial toxins, such as cholera toxin, enter cells by endocytosis and travel retrogradely through the secretory pathway all the way to the ER; here, the toxins
camouflage themselves as misfolded proteins and exit through the proteinconducting channel in the ER membrane into the cytosol, where they inactivate their target molecules and thus kill the cell. Can a virus do the same thing? Richards et al. [1] suggest that this could be the case. The authors have investigated the effects of incubation at 20°C and agents interfering with trafficking mediated by coatomer (COP) I- and II-coated vesicles on SV40 infection and cholera toxin toxicity, and in all cases, both processes are equally affected. This suggests that cholera toxin and SV40 do indeed use the same route to the ER, and that this route includes early endosomes, the trans-Golgi network and the Golgi complex itself. Intriguingly, the authors found that a modified dipeptide, which blocks cholera toxin poisoning at a post-Golgi step, also prevents SV40 infection. Incubation with the peptide depletes intracellular Ca2+ stores, and it has been suggested that it directly affect the channels that mediate cholera toxin transport from the ER to the cytosol. So how does a virus resemble a toxin or a misfolded protein? And if it really uses this
exit from the ER, how does SV40 with a diameter of 45 nm make it through the protein-translocation channel which, even by the most generous estimate, has a maximal opening of 8 nm? There is some evidence that at the plasma membrane SV40 can ‘recruit’ caveolin to surround the viral particles before entry. Perhaps it can do the same with protein-translocation channel subunits and generate a giant pore in the ER membrane. The subdomains of the ER in which the virus accumulates are continuous with the rough ER, but not studded with ribosomes, which primarily bind to channels engaged in protein translocation. So either this region of the ER is devoid of protein-translocation channels or the channels in this region have all become SV40 exits. Time and more experimentation will tell. 1 Richards, A.A. et al. (2002) Inhibitors of COP-mediated transport and cholera toxin action inhibit Simian Virus 40 infection. Mol. Biol. Cell 13, 1750–1764
Karin Römisch [email protected]
mRNA decay: the big picture The cell can precisely regulate expression of any given gene by controlling the rates of transcription and mRNA decay. Although mRNA decay rates can differ by a factor of >100, relatively little is known about the global regulation of this process. Now, Wang et al. [1] have started to redress this imbalance by undertaking a global profiling of mRNA turnover in yeast. They found that mRNA decay rates did not generally correlate with any obvious molecular characteristic but instead seemed to relate to the physiological function of the corresponding gene product. The method adopted by Wang et al. was to combine a well-characterized global transcriptional shut-off assay with DNA microarray analyses. A yeast strain containing a temperature-sensitive allele encoding RNA polymerase II (RNA Pol II) was first grown at 24 °C and then shifted to a restrictive temperature for RNA Pol II and http://tibs.trends.com
samples taken at intervals. The lack of RNA Pol II activity at the elevated temperature meant that changes in steady-state mRNA levels would be mainly determined by specific rates of mRNA decay. RNA was extracted from these samples and microarray analysis used to quantitate the levels of 4687 individual mRNAs at each time point and these data used to calculate half-lives for individual species. A parallel series of experiments provided support for a general mechanism of mRNA decay preceded by shortening of poly(A) tails. The rates observed by Wang et al. varied by a factor of >30, confirming the importance of decay in controlling mRNA turnover during gene expression. However, no relationship could be found between these rates and various factors that had previously been suggested to contribute to decay, such as abundance, ORF size or codon usage. Instead, similarities were
found in decay rates for mRNAs encoding proteins within stoichiometric complexes. For example, 13 of the mRNAs corresponding to the 14 proteins of the 20S proteasome have closely matched decay rates and in the most striking example, 126 of the 131 ribosomal protein-encoding mRNAs analysed have closely clustered decay rates (tellingly, four of the five exceptions are unusual in not being targets of the transcription factor Rap1). The robustness of the correlation between decay rates was supported by a more detailed statistical analysis of 95 protein complexes. Beyond these tightly clustered decay rates associated with complexes, the authors also found broad correlations for mRNAs encoding proteins within functional groups. For example, mRNAs encoding metabolic enzymes were generally long-lived, whereas those associated with transient processes such as mating turned over rapidly.
0968-0004/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.