Один из механизмов перехвата управления
Ученые из Англии выяснили, что один из вспомогательных белков коронавиурса САРС2, а именно ORF6, не являясь структурным белком или ферментом, в то же время очень важен для настройки размножения вируса в клетке. Этот белок подавляет продукцию клеткой ее собственных белков, путем блокирования входа и выхода мессенжерных мРНК из ядра клетки, включая и такие регуляторные мРНК, которые важны для запуска внутриклеточного механизма защиты и врожденного иммунитета.
ORF6 нарушает работу фактора экспорта мРНК, Rae1 (белок, связывающийся с одноцепочечной РНК, считанной с ДНК, и помогающий ей проходить через поры в мембране ядра) и нуклеопорина Nup98(косвенно). Из-за этого клетка не может вырабатывать свои белки (статья прошлого года) , нужные ей для нормального функционирования, но и защищаться - тоже. Потому что заблокированными оказываются и сигнальный путь STAT1/2, и мРНК, "считанные" в ядре после сигналов от внутренних противовирусных факторов IRF1 и RIG-I (специальные внутриклеточные рецепторные и активаторные белки, реагирующие на вирусы, они помогают запустить процесс в интерфероновых генах, чтобы клетка ответила своевременной выработкой интерферона). Не вышла мРНК (для синетеза белка)- не заработала рибосома, не выпустился нужный белок, так что и вирус не заблокировали внутри, и сигнал другим клеткам не послали.. И, поскольку не выходят клеточные мРНК, чтоб загружать рибосомы на выпуск белка, то вирус их использует для того чтобы производить белки для себя, на основе своей РНК.
Так что, блокируя экспорт из ядра мРНК, вирус в первую очередь останавливает врожденный иммунный ответ клетки, и переключает "клеточную машинерию" с выпуска необходимых клетке белков - на вирусные.
[Spoiler (click to open)]
SARS-CoV-2 is the virus responsible for the current COVID-19 pandemic. Coronaviruses, like SARS-CoV-2, replicate their genome in the cytoplasm of the host cell by hijacking the cellular machinery. SARS-CoV-2 replicates in the epithelial cells of the respiratory tract causing extensive respiratory symptoms, with severe cases leading to mortality [1-4]. SARS-CoV-2 expresses four structural proteins, spike (S), membrane (M), envelope (E) and nucleoprotein (N) and 16 non-structural proteins (nsps), as well as nine predicted accessory proteins; ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c and ORF10. Although these are not required for in vitro replication, they are thought to modulate the host cell environment to favour viral replication. In this work, we show that the ORF6 accessory protein can supress cellular protein production by blocking mRNA nuclear export through interacting with the cellular protein Rae1, a known mRNA export factor. We also investigated which cellular mRNAs were retained in the nucleus when ORF6 was overexpressed. Interestingly, we found that ORF6 inhibited the export of many different mRNAs, including those encoding antiviral factors, like IRF1 and RIG-I, even in the absence of stimulation by interferon. Importantly, we found that the export of these mRNAs was similarly affected in the context of SARS-CoV-2 infection. Therefore, we believe we have identified a novel mechanism that SARS-CoV-2 uses to suppress antiviral responses in order to make the cell more permissive to infection. SARS-CoV-2 shares a high degree of homology with the etiological agent of the 2003 SARS outbreak, SARS-CoV (hereafter known as SARS-CoV-1). Indeed, the accessory proteins of these two betacoronaviruses have high homology, with ORF3b and ORF6 being the most divergent. ORF6 from SARS-CoV-1 is a 7.3 kDa protein, which has been reported to inhibit IFN signalling by disrupting the nuclear import of STAT1/STAT2 and IRF3 [8, 9]. This is dependent on ORF6 interacting with the cellular Rae1-Nup98 complex [10], which spans the nuclear envelope, providing a gateway between the nucleus and cytoplasm, and helps shuttle proteins and mRNA between the two compartments [11, 12]. Other viruses have also been shown to disrupt this nuclear export pathway to aid viral propagation.As coronaviruses transcribe their genome in the cytoplasm, they are not dependent on nuclear export function for viral replication. Indeed, NSP1 proteins from coronaviruses have been shown to inhibit the NXF1 mRNA export pathway, potentially disrupting the expression of a range of cellular proteins [17].Given the potential importance of the SARS-CoV-2 accessory proteins we set out to investigate their functions. Strikingly, we found that ORF6 inhibited protein production and we show that in addition to disrupting STAT1/2 import, ORF6 can also block the export of cellular mRNA from the nucleus into the cytoplasm, effectively blocking protein translation. In agreement with other very recent studies of SARS-CoV-2 ORF6, this is also dependent on interaction with the Nup98-Rae1 complex [10, 18, 19]. Here, we utilise cellular fractionation and mRNAseq to identify the mRNA species blocked by ORF6. We show that ORF6 inhibits export of a broad range of mRNAs, in particular several interferon-upregulated genes that encode antiviral factors, including RIG-I and IRF1. This shows that SARS-CoV-2 ORF6 can disrupt host cell innate immune signalling by blocking mRNA nuclear export and suggests that ORF6 may function to inhibit the earliest stages of innate signalling by downregulating expression of pathogen recognition receptors (PRRs) and antiviral transcription factors, such as IRF1. ORF6 has been reported to dislocate Rae1 and Nup98 from the NPC, but whether this mislocalisation causes the mRNA export block or is a consequence of mRNA export disruption is not known [19]. Blocking cellular mRNA nuclear export, thereby reducing translation of cellular proteins, would be advantageous for viral replication as SARS-CoV-2 replicates in the cytoplasm and would therefore be able to exploit the limited cellular translational machinery in favour of viral translation (Fig 6A). In addition, by blocking mRNA export, ORF6 can also affect other cellular processes beyond export, as inhibiting the translation of key cellular proteins will modulate their downstream processes. Excitingly, using RNAseq, we have revealed that ORF6 can prevent the nuclear export of mRNAs encoding antiviral factors (Fig 4), both after IFN treatment, for example IFITM2 and ZNFX1, and more provocatively, in untreated cells. This implies that ORF6 can modulate the basal, steady state level of immunity in cells. Indeed, DDX58(RIG-I) and ZNFX1, which were enriched in the nucleus following both ORF6 expression in untreated cells (Fig 4) and during SARS-CoV-2 infection (Fig 5), are key pattern recognition receptors (PRRs) that detect viral dsRNA [23, 24]. ZNFX1 has been postulated to be involved in the very early stages of IFN signalling, as one of the earliest viral sensors, due to its higher constitutive expression compared to both MDA5 and RIG-I [23]. Downregulation of these sensors would prevent the induction of an antiviral state and allow SARS-CoV-2 to replicate undetected.
The constitutively expressed transcription factor, IRF1, which helps regulate basal expression of antiviral factors like BST2 and RIG-I [25, 26] was also targeted by ORF6. Inhibiting IRF1 mRNA export would therefore have major consequences for the induction of innate immunity [16, 29, 30]. Interestingly, ORF6 from SARS-CoV-1 is thought to be packaged into virions and, thus, the protein is present in the cell as soon as virions infect. Incoming ORF6 could therefore downregulate PRRs before viral replication generates dsRNA intermediates, impeding the earliest antiviral warning system whilst also restricting the expression of further antiviral factors that should induce an antiviral state in the cell [31, 32].
ORF6 нарушает работу фактора экспорта мРНК, Rae1 (белок, связывающийся с одноцепочечной РНК, считанной с ДНК, и помогающий ей проходить через поры в мембране ядра) и нуклеопорина Nup98(косвенно). Из-за этого клетка не может вырабатывать свои белки (статья прошлого года) , нужные ей для нормального функционирования, но и защищаться - тоже. Потому что заблокированными оказываются и сигнальный путь STAT1/2, и мРНК, "считанные" в ядре после сигналов от внутренних противовирусных факторов IRF1 и RIG-I (специальные внутриклеточные рецепторные и активаторные белки, реагирующие на вирусы, они помогают запустить процесс в интерфероновых генах, чтобы клетка ответила своевременной выработкой интерферона). Не вышла мРНК (для синетеза белка)- не заработала рибосома, не выпустился нужный белок, так что и вирус не заблокировали внутри, и сигнал другим клеткам не послали.. И, поскольку не выходят клеточные мРНК, чтоб загружать рибосомы на выпуск белка, то вирус их использует для того чтобы производить белки для себя, на основе своей РНК.
Так что, блокируя экспорт из ядра мРНК, вирус в первую очередь останавливает врожденный иммунный ответ клетки, и переключает "клеточную машинерию" с выпуска необходимых клетке белков - на вирусные.
[Spoiler (click to open)]
SARS-CoV-2 is the virus responsible for the current COVID-19 pandemic. Coronaviruses, like SARS-CoV-2, replicate their genome in the cytoplasm of the host cell by hijacking the cellular machinery. SARS-CoV-2 replicates in the epithelial cells of the respiratory tract causing extensive respiratory symptoms, with severe cases leading to mortality [1-4]. SARS-CoV-2 expresses four structural proteins, spike (S), membrane (M), envelope (E) and nucleoprotein (N) and 16 non-structural proteins (nsps), as well as nine predicted accessory proteins; ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c and ORF10. Although these are not required for in vitro replication, they are thought to modulate the host cell environment to favour viral replication. In this work, we show that the ORF6 accessory protein can supress cellular protein production by blocking mRNA nuclear export through interacting with the cellular protein Rae1, a known mRNA export factor. We also investigated which cellular mRNAs were retained in the nucleus when ORF6 was overexpressed. Interestingly, we found that ORF6 inhibited the export of many different mRNAs, including those encoding antiviral factors, like IRF1 and RIG-I, even in the absence of stimulation by interferon. Importantly, we found that the export of these mRNAs was similarly affected in the context of SARS-CoV-2 infection. Therefore, we believe we have identified a novel mechanism that SARS-CoV-2 uses to suppress antiviral responses in order to make the cell more permissive to infection. SARS-CoV-2 shares a high degree of homology with the etiological agent of the 2003 SARS outbreak, SARS-CoV (hereafter known as SARS-CoV-1). Indeed, the accessory proteins of these two betacoronaviruses have high homology, with ORF3b and ORF6 being the most divergent. ORF6 from SARS-CoV-1 is a 7.3 kDa protein, which has been reported to inhibit IFN signalling by disrupting the nuclear import of STAT1/STAT2 and IRF3 [8, 9]. This is dependent on ORF6 interacting with the cellular Rae1-Nup98 complex [10], which spans the nuclear envelope, providing a gateway between the nucleus and cytoplasm, and helps shuttle proteins and mRNA between the two compartments [11, 12]. Other viruses have also been shown to disrupt this nuclear export pathway to aid viral propagation.As coronaviruses transcribe their genome in the cytoplasm, they are not dependent on nuclear export function for viral replication. Indeed, NSP1 proteins from coronaviruses have been shown to inhibit the NXF1 mRNA export pathway, potentially disrupting the expression of a range of cellular proteins [17].Given the potential importance of the SARS-CoV-2 accessory proteins we set out to investigate their functions. Strikingly, we found that ORF6 inhibited protein production and we show that in addition to disrupting STAT1/2 import, ORF6 can also block the export of cellular mRNA from the nucleus into the cytoplasm, effectively blocking protein translation. In agreement with other very recent studies of SARS-CoV-2 ORF6, this is also dependent on interaction with the Nup98-Rae1 complex [10, 18, 19]. Here, we utilise cellular fractionation and mRNAseq to identify the mRNA species blocked by ORF6. We show that ORF6 inhibits export of a broad range of mRNAs, in particular several interferon-upregulated genes that encode antiviral factors, including RIG-I and IRF1. This shows that SARS-CoV-2 ORF6 can disrupt host cell innate immune signalling by blocking mRNA nuclear export and suggests that ORF6 may function to inhibit the earliest stages of innate signalling by downregulating expression of pathogen recognition receptors (PRRs) and antiviral transcription factors, such as IRF1. ORF6 has been reported to dislocate Rae1 and Nup98 from the NPC, but whether this mislocalisation causes the mRNA export block or is a consequence of mRNA export disruption is not known [19]. Blocking cellular mRNA nuclear export, thereby reducing translation of cellular proteins, would be advantageous for viral replication as SARS-CoV-2 replicates in the cytoplasm and would therefore be able to exploit the limited cellular translational machinery in favour of viral translation (Fig 6A). In addition, by blocking mRNA export, ORF6 can also affect other cellular processes beyond export, as inhibiting the translation of key cellular proteins will modulate their downstream processes. Excitingly, using RNAseq, we have revealed that ORF6 can prevent the nuclear export of mRNAs encoding antiviral factors (Fig 4), both after IFN treatment, for example IFITM2 and ZNFX1, and more provocatively, in untreated cells. This implies that ORF6 can modulate the basal, steady state level of immunity in cells. Indeed, DDX58(RIG-I) and ZNFX1, which were enriched in the nucleus following both ORF6 expression in untreated cells (Fig 4) and during SARS-CoV-2 infection (Fig 5), are key pattern recognition receptors (PRRs) that detect viral dsRNA [23, 24]. ZNFX1 has been postulated to be involved in the very early stages of IFN signalling, as one of the earliest viral sensors, due to its higher constitutive expression compared to both MDA5 and RIG-I [23]. Downregulation of these sensors would prevent the induction of an antiviral state and allow SARS-CoV-2 to replicate undetected.
The constitutively expressed transcription factor, IRF1, which helps regulate basal expression of antiviral factors like BST2 and RIG-I [25, 26] was also targeted by ORF6. Inhibiting IRF1 mRNA export would therefore have major consequences for the induction of innate immunity [16, 29, 30]. Interestingly, ORF6 from SARS-CoV-1 is thought to be packaged into virions and, thus, the protein is present in the cell as soon as virions infect. Incoming ORF6 could therefore downregulate PRRs before viral replication generates dsRNA intermediates, impeding the earliest antiviral warning system whilst also restricting the expression of further antiviral factors that should induce an antiviral state in the cell [31, 32].