Reverse Genetics Demonstrates that Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Essential for Replication in Cell Culture

Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.     

The Processing of Human Rhomboid Intramembrane Serine Protease RHBDL2 Is Required for Its Proteolytic Activity

RHBDL2, a human homolog of the rhomboids, belongs to a unique class of serine intramembrane proteases; little is known about its function and regulation. Here, we show that RHBDL2 is produced as a proenzyme and that the processing of RHBDL2 is required for its cellular protease activity. The processing of RHBDL2 was shown by both Western blot and immunofluorescence analysis. We have demonstrated that a highly conserved Arg residue on loop 1 of RHBDL2 plays a critical role in the activation of the proenzyme. The activation of RHBDL2 is catalyzed by a protease that is sensitive to a class of sulfonamide compounds. Furthermore, endogenous RHBDL2 exists as the processed form and treatment of cells with a sulfonamide inhibitor led to an accumulation of the full length of RHBDL2. Therefore, this study has demonstrated that RHBDL2 activity is regulated by proenzyme activation, revealed a role for the conserved WR residues in loop 1 in RHBDL2 activity, and provided critical insights into the regulation and function of this human rhomboid protease.     

Proteolytic Cleavage of Human Acid-sensing Ion Channel 1 by the Serine Protease Matriptase

Acid-sensing ion channel 1 (ASIC1) is a H⁺-gated channel of the amiloride-sensitive epithelial Na⁺ channel (ENaC)/degenerin family. ASIC1 is expressed mostly in the central and peripheral nervous system neurons. ENaC and ASIC function is regulated by several serine proteases. The type II transmembrane serine protease matriptase activates the prototypical αβγENaC channel, but we found that matriptase is expressed in glioma cells and its expression is higher in glioma compared with normal astrocytes. Therefore, the goal of this study was to test the hypothesis that matriptase regulates ASIC1 function. Matriptase decreased the acid-activated ASIC1 current as measured by two-electrode voltage clamp in Xenopus oocytes and cleaved ASIC1 expressed in oocytes or CHO K1 cells. Inactive S805A matriptase had no effect on either the current or the cleavage of ASIC1. The effect of matriptase on ASIC1 was specific, because it did not affect the function of ASIC2 and no matriptase-specific ASIC2 fragments were detected in oocytes or in CHO cells. Three matriptase recognition sites were identified in ASIC1 (Arg-145, Lys-185, and Lys-384). Site-directed mutagenesis of these sites prevented matriptase cleavage of ASIC1. Our results show that matriptase is expressed in glioma cells and that matriptase specifically cleaves ASIC1 in heterologous expression systems.     

Reverse Genetics Identifies the Product of Open Reading Frame 4 as an Essential Particle Assembly Factor of Nyamanini Virus

We established a reverse genetics system for Nyamanini virus (NYMV) and recovered green fluorescent protein (GFP)-expressing virus from full-length cDNA. Using this technology, we assessed the functions of two poorly characterized viral genes. NYMV lacking open reading frame 2 (ORF2) could not be rescued, whereas virus lacking ORF4 was replication competent. ORF4-deficient NYMV readily established a persisting noncytolytic infection but failed to produce infectious viral particles, supporting the view that ORF4 represents an essential factor for NYMV particle assembly.     

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5''s conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication.     

Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser₄₀₁₄ (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser₄₀₁₄. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn₄₂₀₈, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.Gene 1 of mouse hepatitis virus (MHV) A59 encodes a fusion polyprotein with a predicted mass of 803 kDa (2, 10, 15). Expression of the entire polyprotein of gene 1 requires translation of two overlapping open reading frames (ORFs), 1a and 1b. Since these ORFs are in different reading frames, ORF 1b can be expressed only if a ribosomal frameshift occurs at the end of ORF 1a (4, 5, 21). The ORF 1a portion of gene 1 encodes two experimentally confirmed proteinases, papain-like proteinase 1 (PLP-1) and 3C-like proteinase (3CLpro), as well as an additional proteinase motif, PLP-2, for which no activity has yet been identified (1, 15). The MHV 3CLpro has been shown to autoproteolytically liberate itself from the nascent polyprotein in vitro and in virus-infected cells (in cyto) (18, 19). Eleven cleavage sites have been predicted to be cleaved by 3CLpro, 10 of which have a dipeptide consisting of Gln at position 1 (P1) and Ser, Asp, Gly, or Cys at P1′ (15) (Fig. ​(Fig.1).1). The putative cleavage sites are conserved among the four sequenced coronaviruses and are generally located within the polyprotein and at the putative Q_(S,A,G) dipeptide cleavage site motif (where the underscore indicates the site of cleavage). Six of the predicted MHV 3CLpro cleavage sites are located in a 1,120-amino-acid (aa) region starting at 3CLpro and ending at the carboxy terminus of the ORF 1a polyprotein (aa 3334 to 4454). This region is comprised of 3CLpro as well as a region of predominantly hydrophobic residues between aa 3636 and 3921 (MP-2), a region of unknown function between aa 3922 and 4317, and the putative growth factor-like domain extending from aa 4318 to 4454 (GFL). We were particularly interested in the 532-aa region from the carboxy terminus of the MP-2 domain to the end of GFL, since there are four predicted 3CLpro cleavage sites within this small area and no functions have been proposed for these domains. Open in a separate windowFIG. 1MHV gene 1 organization and putative 3CLpro cleavage sites. The diagram shows the organization of the 22-kb gene 1 of the MHV 32-kb RNA. The locations of the PLP-1 and PLP-2 domains, the MP-1 and MP-2 hydrophobic domains, 3CLpro, the GFL domain, RNA-dependent RNA polymerase (POL), and helicase (HEL) are shown as shaded boxes. Locations of predicted MHV 3CLpro cleavage sites are numbered below the diagram. KR, Lys-Arg dipeptide also proposed as a 3CLpro cleavage site (15). The dots denote the confirmed cleavage sites flanking 3CLpro in the polyprotein. The ∗ indicates the Q_N₄₂₀₈ cleavage site identified and described in this paper. The sequences surrounding the confirmed or putative MHV 3CLpro cleavage sites (denoted by MHV) are aligned with the deduced amino acid sequences of HCV 229E (229E) (11), IBV (3), and TGEV (9). Alignments were performed with MacVector version 6.01.In this study we used a specific antiserum to identify a 22-kDa protein from MHV A59-infected cells that is processed from the region of the ORF 1a polyprotein between MP-2 and the end of ORF 1a (p1a-22). We have shown that 3CLpro is responsible for cleaving this protein at an amino-terminal Gln_Ser site that was previously predicted to be a cleavage site for the proteinase. We also have identified a new cleavage site at the carboxy terminus of the 22-kDa protein that does not conform to the canonical Gln_(Ser,Ala,Gly) motif. Together these results confirm that 3CLpro is responsible for processing at the carboxy-terminal region of the MHV ORF 1a polyprotein.     

Exchange of the Coronavirus Replicase Polyprotein Cleavage Sites Alters Protease Specificity and Processing

Coronavirus nonstructural proteins 1 to 3 are processed by one or two papain-like proteases (PLP1 and PLP2) at specific cleavage sites (CS1 to -3). Murine hepatitis virus (MHV) PLP2 and orthologs recognize and cleave at a position following a p4-Leu-X-Gly-Gly-p1 tetrapeptide, but it is unknown whether these residues are sufficient to result in processing by PLP2 at sites normally cleaved by PLP1. We demonstrate that exchange of CS1 and/or CS2 with the CS3 p4-p1 amino acids in engineered MHV mutants switches specificity from PLP1 to PLP2 at CS2, but not at CS1, and results in altered protein processing and virus replication. Thus, the p4-p1 residues are necessary for PLP2 processing but require a specific protein or cleavage site context for optimal PLP recognition and cleavage.Coronaviruses are positive-strand RNA viruses that translate their first open reading frames (ORF1a and ORF1b) into polyproteins that are processed by viral proteases into intermediate and mature nonstructural proteins (nsp1 to -16) (Fig. ​(Fig.11 A) (4, 7, 17, 20). nsp1, -2, and -3 are liberated at cleavage sites (CSs) between nsp1-2 (CS1), nsp2-3 (CS2), and nsp3-4 (CS3) by one or two papain-like protease (PLP) activities encoded within nsp3 (1, 2, 12, 13, 15) (Fig. ​(Fig.1B).1B). Murine hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E) use two PLPs (PLP1 and PLP2) to process at CS1 to -3, while severe acute respiratory syndrome coronavirus (SARS-CoV) and avian infectious bronchitis virus (IBV) use a single PLP each (PLpro and PLP2, respectively) (10, 20, 25, 26). The factors determining the evolution and use of one versus two PLPs by different coronaviruses for processing of nsp1, -2, and -3 are unknown. Mutations at MHV CSs or within PLP1 alter replication and protein processing in surprising ways (8, 13). Loss of processing at MHV CS1 and CS2 by CS deletion or mutation results in changes in the timing and extent of virus replication. Inactivation of MHV PLP1 is more detrimental for virus replication than deletion of CS1 and CS2 or than inactivation of PLP1 combined with the CS deletions, even though not all of the mutant viruses process at CS1 or CS2 or display similar protein processing phenotypes. In contrast to MHV results, the HCoV-229E PLP1 and PLP2 have both been shown to process at CS1 and CS2, albeit at different efficiencies (Fig. ​(Fig.1B)1B) (24). Finally, the single SARS-CoV PLP2 homolog (PLpro) mediates efficient processing at CS1 to -3, each of which has an upstream position 4-Leu-X-Gly-Gly-position 1 (p4-LXGG-p1) amino acid motif implicated in PLpro processing (10, 16, 18). MHV possesses a p4-LXGG-p1 sequence only at CS3 and is cleaved by PLP2. These results suggest that p4-LXGG-p1 may be the critical determinant of recognition by PLP2/PLpro, but this hypothesis has not been tested in studies of replicating virus. Thus, it remains unknown whether the differences in PLP/CS recognition and processing are determined by the proximal p4-p1 residues (22).Open in a separate windowFIG. 1.MHV replicase organization, coronavirus PLP-mediated processing, and experimental design of cleavage site replacement viruses. (A) ORF1 of MHV genome RNA is shown, with overlapping ORF1a and ORF1b. The ORF1ab polyprotein is shown with nonstructural proteins (nsp1 to -16) indicated by vertical lines and numbers. Viral papain-like protease domains in nsp3 are shown as a white box containing black letters (PLP1) and a black box containing white letters (PLP2), and the nsp5 protease (3CLpro) is indicated as a gray box with a white number. Cleavage sites for PLP1 (CS1 and CS2 [shown as white arrowheads]), PLP2 (CS3 [shown as a black arrowhead]), and nsp5 (CS4 to -14 [shown as gray arrowheads]) are indicated. (B) The organization of nsp1 to nsp4 is shown for representative coronaviruses. PLPs are indicated, with the hatched box in IBV indicating a probable catalytically inactive remnant of PLP1. Processing events that were confirmed as occurring in vitro or during infection are shown by arrows with solid lines and large arrowheads, indicating single or dominant protease activity. The dashed lines and small arrowheads indicate minor or secondary cleavage activities. The CS amino acid sequences from position 4 (p4) to p1′ are shown for each CS, with a space and arrow representing the site of proteolytic processing. (C) The CS substitution viruses were engineered to replace the original CS amino acid sequences at CS1 and/or CS2 with that of the CS3 amino acid sequence p4-LKGG-p1. Both CS substitutions were also engineered into a catalytically inactive PLP1 (P1ko) background. PLPs are shown as numbers in boxes within nsp3. Engineered catalytically inactivated PLP1 is shown as a hatched box. Arrowheads indicate cleavage events of the WT virus and are linked to the enzyme predicted to mediate processing at the CS, as indicated by white boxes containing black characters (PLP1) or black boxes containing white characters (PLP2). The p4 through p1 amino acid residues for each CS are shown below each diagram. White and black vertical bars show the respective predicted PLP1 and PLP2 cleavage sites. Engineered substitutions are indicated in bold characters. Asterisks indicate engineered mutant genomes that could not be recovered as infectious virus.In this study, we used MHV as a model to test whether PLP/CS specificities could be switched by an exchange of CS amino acid sequences and to determine the impact of CS exchange on protein processing and virus replication. Replacement of the CS3 p4-LKGG-p1 at CS2, but not at CS1, was sufficient for a switch in protease specificity from PLP1 to PLP2. Some combinations of CS exchange could not be recovered with inactive PLP1, and recovered mutant viruses had altered protein processing and/or impaired growth compared to the wild type (WT). The results confirm that p4-LXGG-p1 amino acid sequences are necessary determinants of cleavage by PLP2 but also indicate that a larger cleavage site or a different protein context is required for efficient recognition and processing. Finally, the results support the conclusion that complex relationships with respect to the timing and extent of PLP/CS interactions are essential for successful replication and, likely, for virus fitness.     

Interaction between the Human Cytomegalovirus Tegument Proteins UL94 and UL99 Is Essential for Virus Replication

Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood, especially with respect to the cytoplasmic phase of assembly, during which the majority of the tegument is acquired and final envelopment occurs. These processes occur at a unique cytoplasmic structure called the assembly complex, which is formed through a reorganization of the cellular secretory apparatus. The HCMV tegument protein UL99 (pp28) is essential for viral replication at the stage of secondary envelopment. We previously demonstrated that UL99 interacts with the essential tegument protein UL94 in infected cells as well as in the absence of other viral proteins. Here we show that UL94 and UL99 alter each other's localization and that UL99 stabilizes UL94 in a binding-dependent manner. We have mapped the interaction between UL94 and UL99 to identify the amino acids of each protein that are required for their interaction. Mutation of these amino acids in the context of the viral genome demonstrates that HCMV is completely defective for replication in the absence of the interaction between UL94 and UL99. Further, we demonstrate that in the absence of their interaction, both UL94 and UL99 exhibit aberrant localization and do not accumulate at the assembly complex during infection. Taken together, our data suggest that the interaction between UL94 and UL99 is essential for the proper localization of each protein to the assembly complex and thus for the production of infectious virus.     

EWSR1 Binds the Hepatitis C Virus cis-Acting Replication Element and Is Required for Efficient Viral Replication

The hepatitis C virus (HCV) genome contains numerous RNA elements that are required for its replication. Most of the identified RNA structures are located within the 5′ and 3′ untranslated regions (UTRs). One prominent RNA structure, termed the cis-acting replication element (CRE), is located within the NS5B coding region. Mutation of part of the CRE, the 5BSL3.2 stem-loop, impairs HCV RNA replication. This loop has been implicated in a kissing interaction with a complementary stem-loop structure in the 3′ UTR. Although it is clear that this interaction is required for viral replication, the function of the interaction, and its regulation are unknown. In order to gain insight into the CRE function, we isolated cellular proteins that preferentially bind the CRE and identified them using mass spectrometry. This approach identified EWSR1 as a CRE-binding protein. Silencing EWSR1 expression impairs HCV replication and infectious virus production but not translation. While EWRS1 is a shuttling protein that is extensively nuclear in hepatocytes, substantial amounts of EWSR1 localize to the cytosol in HCV-infected cells and colocalize with sites of HCV replication. A subset of EWRS1 translocates into detergent-resistant membrane fractions, which contain the viral replicase proteins, in cells with replicating HCV. EWSR1 directly binds the CRE, and this is dependent on the intact CRE structure. Finally, EWSR1 preferentially interacts with the CRE in the absence of the kissing interaction. This study implicates EWSR1 as a novel modulator of CRE function in HCV replication.