- Email: [email protected]
No-Go’ing Back: Co-opting RVB-2 to Control HIV-1 Gene Expression and Immune Response
TIMI 1237 No. of Pages 3
Spotlight
Production of infectious HIV-1 particles requires viral envelope (Env) glycoprotein incorporation. Although, the precise mechanism remains elusive, interaction between Env and the matrix (MA) domain of Gag plays a central role. Work by Mu and colleagues demonstrates how the Env–MA interaction regulates gag mRNA stability and Gag expression levels.
multiple functions. The authors elegantly demonstrate, in a logical series of experiments, that RVB-2 interacts with the MA domain of Gag and the 50 untranslated region (UTR) of the Gag genomic mRNA (gag mRNA) to impede translation and activate no-go mRNA decay (NGD, see below). Furthermore, they show that the exposed CTDEnv competes for MA binding and releases gag mRNA from this blockade. Striking a fine balance between Gag and Env expression levels impacts upon virus production and provides support for a role of this regulatory circuit in the timing of virus assembly (Figure 1). Furthermore, RVB-2 mRNA levels correlated, albeit loosely, with viral loads in patients who progressed rapidly to acquired immunodeficiency syndrome (AIDS) or in those who did not (known as long-term non-progressors). This study reveals a mechanism by which HIV-1 produces a maximum amount of infectious virus but only when sufficient levels of Env are present so as to not trigger an early immune response.
Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane. The expression of the structural protein Gag is sufficient to produce virus-like particles but incorporation of the viral envelope (Env) glycoprotein into the lipid bilayer of nascent particles renders HIV-1 infectious. Env is necessary for mediating fusion between viral and cellular membranes during virus entry. The mechanism behind Env incorporation into virus particles remains elusive yet substantial evidence suggests that an interaction between the matrix (MA) domain of Gag and the C-terminal domain of Env (CTDEnv) promotes virus infectivity [1]. The Cell Host & Microbe report by Mu and colleagues uncovers a new mechanism of control of viral gene expression that requires the binding of MA–CTDEnv and involves a novel virus–host interaction between RuvB-like 2 (RVB-2) and HIV-1 Gag [2]. To ensure adequate Gag expression, HIV1 has evolved to co-opt RVB-2, a ubiquitously expressed host protein with
Intimate connections between gene expression phases (transcription, RNA maturation, export, trafficking, translation, and turnover) is the current dogma in post-transcriptional gene regulation [3]. This work underscores again how these processes are likewise closely linked for HIV-1. RNA trafficking, which is coupled to all of these processes, has received enormous attention in the field of HIV-1 research since the discovery that viral RNA nuclear export is regulated by a small regulatory protein named Rev and a constellation of host factors. In the cytoplasm, Rev also impacts on the translatability of the gag mRNA and on its cytoplasmic accumulation (see [4]). Dr. Gao's research group has focused on studying coupled processes in the post-transcriptional regulation of viral gene expression in their examination of the interplay between translational repression and mRNA decay in the context of viral infection. Dr. Gao's latest work underscores how these steps are intimately linked for HIV-1 [2].
No-Go’ing Back: Co-opting RVB-2 to Control HIV-1 Gene Expression and Immune Response Valerie Le Sage,1 Alessandro Cinti,1,2 and Andrew J. Mouland1,2,3,*
It is almost routine now for HIV-1 to hijack and co-opt host cell machineries and this is no exception, except with a twist. Cellular mRNA surveillance machineries [nonsense-mediated decay (NMD), NGD, and non-stop decay (NSD)] are on the lookout for aberrant mRNAs and stalled ribosome elongation complexes [5], which would putatively include those that contain introns like the singly-spliced 4 kb and the unspliced 9 kb gag mRNAs. A comparison to the characterized NGD is made in this paper whereby following ribosome elongation complex stalling on the gag mRNA, host release factors [eRF1/Pelota (in mammals) and eRF3] are engaged to elicit mRNA degradation by 50 decapping and the cleavage by exo/endonucleases [6]. eRF1/Pelota was shown to be important for the RVB-2-mediated degradation of the gag mRNA. Likely, eRF1/Pelota engagement serves as a signal that leads to the release of ribosomes from the gag mRNA and targeting for degradation. It is interesting that HIV-1 succumbs to translation-coupled events such as NGD, as it seems counterintuitive that viral mRNA degradation would be beneficial to virus production. Nevertheless, eRF1/Pelota appears to maintain gag mRNA stability, is a key player in regulating the 20:1 Gag: GagPol stoichiometry, and regulates the packaging of 2 gag mRNAs per progeny virus [7]. This is the first functional report for the involvement of RVB-2 in HIV-1 replication and contributes to our understanding of the control of Gag expression, which was once thought to be unrestricted in an infected cell. It is, however, not the first to intimate function. RVB-2 was shown to interact with Tat, was found in HIV-1 cores, and found to associate with several other HIV-1 proteins (e.g., [8]). These earlier reports on RVB-2 might be in line with the current findings as Tat binds the 50 end of the gag mRNA and remnants of these virus–host interactions might remain as the translation of the gag mRNA is turned on later during assembly.
Trends in Microbiology, September 2015, Vol xx. No. x
1
TIMI 1237 No. of Pages 3
CTD MA
RVB2
Gag
Env
eRF1/Pelota gag mRNA
m7G
gag mRNA
5′UTR
Nucleus
(A)
(C)
Cytoplasm
(B)
m7G
5′UTR
m7G
m7G
5′UTR
5′UTR
Stalled ribosome ‘Bald’ virions
Infecous virus
No-go mRNA decay
gag mRNA cleavage
Figure 1. A Schematic Model of HIV-1 Gag Expression Regulated by the Host Factor RVB-2. Export of the viral genomic mRNA (gag mRNA) from the nucleus (top right) results in the translation of the Gag structural protein, which is regulated by the proportion of RVB-2 and the C-terminal domain of envelope (EnvCTD) (yellow circles) in the cytoplasm. The expression of Gag is sufficient to produce ‘bald’, non-infectious virus particles, which occurs upon depletion of RVB-2 and in the absence of sufficient quantities of the Env protein as shown in (A). A fine balance in Gag expression is orchestrated by RVB-2 (B) through an interaction between the host protein and the nascent matrix (MA) domain of Gag (red circles) and the 50 untranslated region (UTR) of the target gag mRNA. These binding events lead to translational stalling that blocks Gag protein expression and initiates no-go mRNA decay through the recruitment of eRF1/Pelota resulting in gag mRNA cleavage. (C) Upon reaching a threshold of Env protein, the EnvCTD competes with RVB-2 for binding to MA and allows Gag synthesis to proceed and assembly of infectious HIV-1 virions.
This study is comprehensive and elegant in most respects. The burst of Gag expression afforded by RVB-2/CTDEnv– MA competition does not however account for contributions of conformational changes in the 50 UTR of the gag mRNA that occur late in the gene expression phase [9], nor is it entirely consistent with earlier findings on Gag's ability to autoregulate its own synthesis [10]. But, the study serves as a reminder for the multiple fates and roles for the viral Gag protein in the cytoplasm with populations that are regulated and others that appear to be immobile. As Gag is synthesized, multiple controls are exerted by cis-acting sequences in Gag and in the cognate gag mRNA, to which multiple host proteins
2
Trends in Microbiology, September 2015, Vol xx. No. x
bind. Of note, these include several that are involved in host mRNA decay and surveillance machineries including NGD (RVB-2), NMD (UPF1) and Staufen1mediated mRNA decay, SMD (UPF1, Staufen1), with in common host factors such as eRF1/Pelota contributing to the regulation. While resistant to the last two decay machineries, at least in the classical way [5], the recruitment of eRF1/Pelota and perhaps other players involved in the clearance of mRNA remains an important finding that currently stands out in the field. Several other mRNA decay-associated factors such as DDX6, Ago2, and Mov10, for example, are recruited by HIV-1 for assembly, which may in fact be a means to subvert them from their
normal mRNA decay functions in host cells. Considering that only a very small percentage of virus is fully infectious as a number of concerted events and viral protein stoichiometries must be satisfied (e.g., [7]), regulation by RVB-2 likely contributes to upping the number of infectious virus. Now, expression of Env from its cognate mRNAs is known to be post-transcriptionally regulated, but is there an equally elusive way by which Env expression is ensured? The importance of this mechanism may be HIV-1-specific as HIV-1 has indeed evolved several unique strategies that lay claim to its highly pathogenic potential. Evidence is presented on the
TIMI 1237 No. of Pages 3
universality of RVB-2 function, but the divergence of Gag, Env, and 50 UTR sequences in the other retroviruses tested [HIV-2, simian immunodeficiency virus (SIV)] tells us that this will necessitate further examination. This study provides an impetus to further our understanding of key post-transcriptional events during HIV-1 replication. It also has important implications in future therapeutic targeting, in providing clues to upping viral clearance by enhancing the immune response to HIV1 and in curing the infection once and for all. Acknowledgments The authors apologize for not being able to refer to other important findings in the field. We thank Ricardo Soto-Rifo (Universidad de Chile) for helpful
suggestions on content. Work from the authors’ laboratory is supported by grants from the Canadian Institutes of Health Research (MOP-38111, MOP56974, and HIG-133050). 1
HIV-1 RNA Trafficking Laboratory, Lady Davis Institute at the Jewish General Hospital, Montréal, Québec, H3T 1E2, Canada 2 Department of Medicine, McGill University, Montréal, Québec, H3A 0G4, Canada 3
Department of Microbiology and Immunology, McGill University, Montréal, Québec, H3A 0G4, Canada *Correspondence: [email protected] (A.J. Mouland). http://dx.doi.org/10.1016/j.tim.2015.08.006 References 1. Cosson, P. (1996) Direct interaction between the envelope and matrix proteins of HIV-1. EMBO J. 15, 5783–5788 2. Mu, X. et al. (2015) HIV-1 exploits the host factor RuvB-like 2 to balance viral protein expression. Cell Host Microbe 18, 233–242
3. Danckwardt, S. et al. (2008) 30 end mRNA processing: molecular mechanisms and implications for health and disease. EMBO J. 27, 482–498 4. Yedavalli, V.S. and Jeang, K.T. (2011) Rev-ing up post-transcriptional HIV-1 RNA expression. RNA Biol. 8, 195–199 5. Ajamian, L. et al. (2008) Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation. RNA 14, 914–927 6. Pisareva, V.P. et al. (2011) Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J. 30, 1804– 1817 7. Chamanian, M. et al. (2013) A cis-acting element in retroviral genomic RNA links Gag-Pol ribosomal frameshifting to selective viral RNA encapsidation. Cell Host Microbe 13, 181–192 8. Jager, S. et al. (2012) Global landscape of HIV-human protein complexes. Nature 481, 365–370 9. Lu, K. et al. (2011) NMR detection of structures in the HIV-1 50 -leader RNA that regulate genome packaging. Science 334, 242–245 10. Anderson, E.C. and Lever, A.M. (2006) Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation. J. Virol. 80, 10478–10486
Trends in Microbiology, September 2015, Vol xx. No. x
3
Spotlight
Production of infectious HIV-1 particles requires viral envelope (Env) glycoprotein incorporation. Although, the precise mechanism remains elusive, interaction between Env and the matrix (MA) domain of Gag plays a central role. Work by Mu and colleagues demonstrates how the Env–MA interaction regulates gag mRNA stability and Gag expression levels.
multiple functions. The authors elegantly demonstrate, in a logical series of experiments, that RVB-2 interacts with the MA domain of Gag and the 50 untranslated region (UTR) of the Gag genomic mRNA (gag mRNA) to impede translation and activate no-go mRNA decay (NGD, see below). Furthermore, they show that the exposed CTDEnv competes for MA binding and releases gag mRNA from this blockade. Striking a fine balance between Gag and Env expression levels impacts upon virus production and provides support for a role of this regulatory circuit in the timing of virus assembly (Figure 1). Furthermore, RVB-2 mRNA levels correlated, albeit loosely, with viral loads in patients who progressed rapidly to acquired immunodeficiency syndrome (AIDS) or in those who did not (known as long-term non-progressors). This study reveals a mechanism by which HIV-1 produces a maximum amount of infectious virus but only when sufficient levels of Env are present so as to not trigger an early immune response.
Human immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane. The expression of the structural protein Gag is sufficient to produce virus-like particles but incorporation of the viral envelope (Env) glycoprotein into the lipid bilayer of nascent particles renders HIV-1 infectious. Env is necessary for mediating fusion between viral and cellular membranes during virus entry. The mechanism behind Env incorporation into virus particles remains elusive yet substantial evidence suggests that an interaction between the matrix (MA) domain of Gag and the C-terminal domain of Env (CTDEnv) promotes virus infectivity [1]. The Cell Host & Microbe report by Mu and colleagues uncovers a new mechanism of control of viral gene expression that requires the binding of MA–CTDEnv and involves a novel virus–host interaction between RuvB-like 2 (RVB-2) and HIV-1 Gag [2]. To ensure adequate Gag expression, HIV1 has evolved to co-opt RVB-2, a ubiquitously expressed host protein with
Intimate connections between gene expression phases (transcription, RNA maturation, export, trafficking, translation, and turnover) is the current dogma in post-transcriptional gene regulation [3]. This work underscores again how these processes are likewise closely linked for HIV-1. RNA trafficking, which is coupled to all of these processes, has received enormous attention in the field of HIV-1 research since the discovery that viral RNA nuclear export is regulated by a small regulatory protein named Rev and a constellation of host factors. In the cytoplasm, Rev also impacts on the translatability of the gag mRNA and on its cytoplasmic accumulation (see [4]). Dr. Gao's research group has focused on studying coupled processes in the post-transcriptional regulation of viral gene expression in their examination of the interplay between translational repression and mRNA decay in the context of viral infection. Dr. Gao's latest work underscores how these steps are intimately linked for HIV-1 [2].
No-Go’ing Back: Co-opting RVB-2 to Control HIV-1 Gene Expression and Immune Response Valerie Le Sage,1 Alessandro Cinti,1,2 and Andrew J. Mouland1,2,3,*
It is almost routine now for HIV-1 to hijack and co-opt host cell machineries and this is no exception, except with a twist. Cellular mRNA surveillance machineries [nonsense-mediated decay (NMD), NGD, and non-stop decay (NSD)] are on the lookout for aberrant mRNAs and stalled ribosome elongation complexes [5], which would putatively include those that contain introns like the singly-spliced 4 kb and the unspliced 9 kb gag mRNAs. A comparison to the characterized NGD is made in this paper whereby following ribosome elongation complex stalling on the gag mRNA, host release factors [eRF1/Pelota (in mammals) and eRF3] are engaged to elicit mRNA degradation by 50 decapping and the cleavage by exo/endonucleases [6]. eRF1/Pelota was shown to be important for the RVB-2-mediated degradation of the gag mRNA. Likely, eRF1/Pelota engagement serves as a signal that leads to the release of ribosomes from the gag mRNA and targeting for degradation. It is interesting that HIV-1 succumbs to translation-coupled events such as NGD, as it seems counterintuitive that viral mRNA degradation would be beneficial to virus production. Nevertheless, eRF1/Pelota appears to maintain gag mRNA stability, is a key player in regulating the 20:1 Gag: GagPol stoichiometry, and regulates the packaging of 2 gag mRNAs per progeny virus [7]. This is the first functional report for the involvement of RVB-2 in HIV-1 replication and contributes to our understanding of the control of Gag expression, which was once thought to be unrestricted in an infected cell. It is, however, not the first to intimate function. RVB-2 was shown to interact with Tat, was found in HIV-1 cores, and found to associate with several other HIV-1 proteins (e.g., [8]). These earlier reports on RVB-2 might be in line with the current findings as Tat binds the 50 end of the gag mRNA and remnants of these virus–host interactions might remain as the translation of the gag mRNA is turned on later during assembly.
Trends in Microbiology, September 2015, Vol xx. No. x
1
TIMI 1237 No. of Pages 3
CTD MA
RVB2
Gag
Env
eRF1/Pelota gag mRNA
m7G
gag mRNA
5′UTR
Nucleus
(A)
(C)
Cytoplasm
(B)
m7G
5′UTR
m7G
m7G
5′UTR
5′UTR
Stalled ribosome ‘Bald’ virions
Infecous virus
No-go mRNA decay
gag mRNA cleavage
Figure 1. A Schematic Model of HIV-1 Gag Expression Regulated by the Host Factor RVB-2. Export of the viral genomic mRNA (gag mRNA) from the nucleus (top right) results in the translation of the Gag structural protein, which is regulated by the proportion of RVB-2 and the C-terminal domain of envelope (EnvCTD) (yellow circles) in the cytoplasm. The expression of Gag is sufficient to produce ‘bald’, non-infectious virus particles, which occurs upon depletion of RVB-2 and in the absence of sufficient quantities of the Env protein as shown in (A). A fine balance in Gag expression is orchestrated by RVB-2 (B) through an interaction between the host protein and the nascent matrix (MA) domain of Gag (red circles) and the 50 untranslated region (UTR) of the target gag mRNA. These binding events lead to translational stalling that blocks Gag protein expression and initiates no-go mRNA decay through the recruitment of eRF1/Pelota resulting in gag mRNA cleavage. (C) Upon reaching a threshold of Env protein, the EnvCTD competes with RVB-2 for binding to MA and allows Gag synthesis to proceed and assembly of infectious HIV-1 virions.
This study is comprehensive and elegant in most respects. The burst of Gag expression afforded by RVB-2/CTDEnv– MA competition does not however account for contributions of conformational changes in the 50 UTR of the gag mRNA that occur late in the gene expression phase [9], nor is it entirely consistent with earlier findings on Gag's ability to autoregulate its own synthesis [10]. But, the study serves as a reminder for the multiple fates and roles for the viral Gag protein in the cytoplasm with populations that are regulated and others that appear to be immobile. As Gag is synthesized, multiple controls are exerted by cis-acting sequences in Gag and in the cognate gag mRNA, to which multiple host proteins
2
Trends in Microbiology, September 2015, Vol xx. No. x
bind. Of note, these include several that are involved in host mRNA decay and surveillance machineries including NGD (RVB-2), NMD (UPF1) and Staufen1mediated mRNA decay, SMD (UPF1, Staufen1), with in common host factors such as eRF1/Pelota contributing to the regulation. While resistant to the last two decay machineries, at least in the classical way [5], the recruitment of eRF1/Pelota and perhaps other players involved in the clearance of mRNA remains an important finding that currently stands out in the field. Several other mRNA decay-associated factors such as DDX6, Ago2, and Mov10, for example, are recruited by HIV-1 for assembly, which may in fact be a means to subvert them from their
normal mRNA decay functions in host cells. Considering that only a very small percentage of virus is fully infectious as a number of concerted events and viral protein stoichiometries must be satisfied (e.g., [7]), regulation by RVB-2 likely contributes to upping the number of infectious virus. Now, expression of Env from its cognate mRNAs is known to be post-transcriptionally regulated, but is there an equally elusive way by which Env expression is ensured? The importance of this mechanism may be HIV-1-specific as HIV-1 has indeed evolved several unique strategies that lay claim to its highly pathogenic potential. Evidence is presented on the
TIMI 1237 No. of Pages 3
universality of RVB-2 function, but the divergence of Gag, Env, and 50 UTR sequences in the other retroviruses tested [HIV-2, simian immunodeficiency virus (SIV)] tells us that this will necessitate further examination. This study provides an impetus to further our understanding of key post-transcriptional events during HIV-1 replication. It also has important implications in future therapeutic targeting, in providing clues to upping viral clearance by enhancing the immune response to HIV1 and in curing the infection once and for all. Acknowledgments The authors apologize for not being able to refer to other important findings in the field. We thank Ricardo Soto-Rifo (Universidad de Chile) for helpful
suggestions on content. Work from the authors’ laboratory is supported by grants from the Canadian Institutes of Health Research (MOP-38111, MOP56974, and HIG-133050). 1
HIV-1 RNA Trafficking Laboratory, Lady Davis Institute at the Jewish General Hospital, Montréal, Québec, H3T 1E2, Canada 2 Department of Medicine, McGill University, Montréal, Québec, H3A 0G4, Canada 3
Department of Microbiology and Immunology, McGill University, Montréal, Québec, H3A 0G4, Canada *Correspondence: [email protected] (A.J. Mouland). http://dx.doi.org/10.1016/j.tim.2015.08.006 References 1. Cosson, P. (1996) Direct interaction between the envelope and matrix proteins of HIV-1. EMBO J. 15, 5783–5788 2. Mu, X. et al. (2015) HIV-1 exploits the host factor RuvB-like 2 to balance viral protein expression. Cell Host Microbe 18, 233–242
3. Danckwardt, S. et al. (2008) 30 end mRNA processing: molecular mechanisms and implications for health and disease. EMBO J. 27, 482–498 4. Yedavalli, V.S. and Jeang, K.T. (2011) Rev-ing up post-transcriptional HIV-1 RNA expression. RNA Biol. 8, 195–199 5. Ajamian, L. et al. (2008) Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation. RNA 14, 914–927 6. Pisareva, V.P. et al. (2011) Dissociation by Pelota, Hbs1 and ABCE1 of mammalian vacant 80S ribosomes and stalled elongation complexes. EMBO J. 30, 1804– 1817 7. Chamanian, M. et al. (2013) A cis-acting element in retroviral genomic RNA links Gag-Pol ribosomal frameshifting to selective viral RNA encapsidation. Cell Host Microbe 13, 181–192 8. Jager, S. et al. (2012) Global landscape of HIV-human protein complexes. Nature 481, 365–370 9. Lu, K. et al. (2011) NMR detection of structures in the HIV-1 50 -leader RNA that regulate genome packaging. Science 334, 242–245 10. Anderson, E.C. and Lever, A.M. (2006) Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation. J. Virol. 80, 10478–10486
Trends in Microbiology, September 2015, Vol xx. No. x
3