The 5' noncoding region of the poliovirus genome contains RNA structures important for re... more The 5' noncoding region of the poliovirus genome contains RNA structures important for replication and translation. Here we show that two closely related cellular poly(rC) binding proteins (PCBP1 and PCBP2) bind to the terminal cloverleaf structure and facilitate the interaction of the viral protein 3CD (the uncleaved precursor of the protease-polymerase). In addition, these cellular proteins bind to stem-loop IV of the internal ribosomal entry site. The proteins are cytoplasmic and largely associated with ribosomes; they appear to dimerize in solution and to form heterodimers when binding to stem-loop IV. Initiation of viral translation in Xenopus oocytes is strongly inhibited by co-injection of specific antibodies directed against PCBP1 or PCBP2, indicating that the poly(rC) binding proteins may facilitate this process. Furthermore, PCPB-depleted HeLa extracts translate poliovirus RNA inefficiently and the activity is partially restored by addition of recombinant PCBP proteins.
The NS2B–NS3 protease complex is essential for the replication of dengue virus, which is the etio... more The NS2B–NS3 protease complex is essential for the replication of dengue virus, which is the etiologic agent of dengue and hemorrhagic fevers, diseases that are a burden for the tropical and subtropical areas of the world. The active form of the NS3 protease linked to the 40 residues of the NS2B cofactor shows highly flexible and disordered region(s) that are responsible for its high propensity to aggregate at the concentrations necessary for NMR spectroscopy studies or for crystallization. Limited proteolysis of this active form of the protease enabled us to obtain a folded and new essential form of the NS2B–NS3 protease complex. We found that the region from residues D50 to E80 of NS2B interacts directly and strongly with the NS3 protease domain. The proteolytic activity of the noncovalently binding complex was determined by a rapid and continuous fluorescence resonance energy transfer activity assay using a depsipeptide substrate. The new protein–cofactor complex obtained, encompassing the NS2B fragment (D50–E80) and the NS3 protease, shows proteolytic activity. The 1H-15N-heteronuclear single quantum coherence spectrum of the isotopically enriched protein complex shows good cross-peak dispersion; this is indicative of a stable folded state. Our results significantly complement the X-ray structure of the NS2B–NS3pro complex published recently. Moreover, these results open the way to performing direct structural and interaction studies in solution on a new active NS2B–NS3pro complex with libraries of substrates and inhibitors in order to identify new drugs that prevent viral polyprotein processing.
Flavivirus genome amplification is a complex process that involves the viral RNA, cellular and vi... more Flavivirus genome amplification is a complex process that involves the viral RNA, cellular and viral proteins, and a sophisticated architecture of cellular membranes induced by viral infection. The viral RNA is not just a passive template; it plays an active role acquiring dynamic tertiary structures during viral replication. RNA synthesis is regulated by cis-acting elements present at the 5′- and 3′-ends of the viral genome. These elements include complementary sequences that mediate genome cyclization through direct RNA–RNA interactions. Studies from many laboratories have provided compelling evidence supporting the notion that a circular conformation of the viral RNA is essential for flavivirus RNA replication. In addition, an RNA element located within the viral 5′UTR has been found to bind the viral polymerase and promote RNA synthesis. In this chapter, we describe viral proteins and RNA structures involved in flavivirus genome amplification and provide working models that explain the need of long-range RNA–RNA interactions during viral RNA synthesis.
We have developed a dengue virus replicon system that can be used to discriminate between transla... more We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3′UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3′UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5′ and 3′ ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3′UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Δ30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3′UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.
Maturation of infectious human immunodeficiency virus type 1 (HIV-1) particles requires proteolyt... more Maturation of infectious human immunodeficiency virus type 1 (HIV-1) particles requires proteolytic cleavage of structural polyproteins by viral protease. Inhibition of protease is a powerful tool for the treatment of HIV infection. Using a well-established phenotypic drug susceptibility assay, we found that sequences outside of the protease gene can modulate the susceptibility to protease inhibitors (PIs). Chimeric viruses carrying p1-p6/p6* sequences from patient isolates in the context of an NL4-3 molecular clone exhibited increased PI susceptibility. Furthermore, this phenotype was associated with a delay in protease autoprocessing in virions and a reduction in replication capacity. We propose that the interplay between protease and the C terminus of Gag is critical for proper protease activity and mismatches between these regions can reduce viral replication and increase drug susceptibility.
The 5' noncoding region of the poliovirus genome contains RNA structures important for re... more The 5' noncoding region of the poliovirus genome contains RNA structures important for replication and translation. Here we show that two closely related cellular poly(rC) binding proteins (PCBP1 and PCBP2) bind to the terminal cloverleaf structure and facilitate the interaction of the viral protein 3CD (the uncleaved precursor of the protease-polymerase). In addition, these cellular proteins bind to stem-loop IV of the internal ribosomal entry site. The proteins are cytoplasmic and largely associated with ribosomes; they appear to dimerize in solution and to form heterodimers when binding to stem-loop IV. Initiation of viral translation in Xenopus oocytes is strongly inhibited by co-injection of specific antibodies directed against PCBP1 or PCBP2, indicating that the poly(rC) binding proteins may facilitate this process. Furthermore, PCPB-depleted HeLa extracts translate poliovirus RNA inefficiently and the activity is partially restored by addition of recombinant PCBP proteins.
The NS2B–NS3 protease complex is essential for the replication of dengue virus, which is the etio... more The NS2B–NS3 protease complex is essential for the replication of dengue virus, which is the etiologic agent of dengue and hemorrhagic fevers, diseases that are a burden for the tropical and subtropical areas of the world. The active form of the NS3 protease linked to the 40 residues of the NS2B cofactor shows highly flexible and disordered region(s) that are responsible for its high propensity to aggregate at the concentrations necessary for NMR spectroscopy studies or for crystallization. Limited proteolysis of this active form of the protease enabled us to obtain a folded and new essential form of the NS2B–NS3 protease complex. We found that the region from residues D50 to E80 of NS2B interacts directly and strongly with the NS3 protease domain. The proteolytic activity of the noncovalently binding complex was determined by a rapid and continuous fluorescence resonance energy transfer activity assay using a depsipeptide substrate. The new protein–cofactor complex obtained, encompassing the NS2B fragment (D50–E80) and the NS3 protease, shows proteolytic activity. The 1H-15N-heteronuclear single quantum coherence spectrum of the isotopically enriched protein complex shows good cross-peak dispersion; this is indicative of a stable folded state. Our results significantly complement the X-ray structure of the NS2B–NS3pro complex published recently. Moreover, these results open the way to performing direct structural and interaction studies in solution on a new active NS2B–NS3pro complex with libraries of substrates and inhibitors in order to identify new drugs that prevent viral polyprotein processing.
Flavivirus genome amplification is a complex process that involves the viral RNA, cellular and vi... more Flavivirus genome amplification is a complex process that involves the viral RNA, cellular and viral proteins, and a sophisticated architecture of cellular membranes induced by viral infection. The viral RNA is not just a passive template; it plays an active role acquiring dynamic tertiary structures during viral replication. RNA synthesis is regulated by cis-acting elements present at the 5′- and 3′-ends of the viral genome. These elements include complementary sequences that mediate genome cyclization through direct RNA–RNA interactions. Studies from many laboratories have provided compelling evidence supporting the notion that a circular conformation of the viral RNA is essential for flavivirus RNA replication. In addition, an RNA element located within the viral 5′UTR has been found to bind the viral polymerase and promote RNA synthesis. In this chapter, we describe viral proteins and RNA structures involved in flavivirus genome amplification and provide working models that explain the need of long-range RNA–RNA interactions during viral RNA synthesis.
We have developed a dengue virus replicon system that can be used to discriminate between transla... more We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3′UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3′UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5′ and 3′ ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3′UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Δ30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3′UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.
Maturation of infectious human immunodeficiency virus type 1 (HIV-1) particles requires proteolyt... more Maturation of infectious human immunodeficiency virus type 1 (HIV-1) particles requires proteolytic cleavage of structural polyproteins by viral protease. Inhibition of protease is a powerful tool for the treatment of HIV infection. Using a well-established phenotypic drug susceptibility assay, we found that sequences outside of the protease gene can modulate the susceptibility to protease inhibitors (PIs). Chimeric viruses carrying p1-p6/p6* sequences from patient isolates in the context of an NL4-3 molecular clone exhibited increased PI susceptibility. Furthermore, this phenotype was associated with a delay in protease autoprocessing in virions and a reduction in replication capacity. We propose that the interplay between protease and the C terminus of Gag is critical for proper protease activity and mismatches between these regions can reduce viral replication and increase drug susceptibility.
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Papers by Andrea Gamarnik