Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity “Arg-like” SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an “Abl-like” low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases.     

CD2-associated Protein (CD2AP) Enhances Casitas B Lineage Lymphoma-3/c (Cbl-3/c)-mediated Ret Isoform-specific Ubiquitination and Degradation via Its Amino-terminal Src Homology 3 Domains

Ret is the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of neuronal growth factors. Upon activation by GDNF, Ret is rapidly polyubiquitinated and degraded. This degradation process is isoform-selective, with the longer Ret51 isoform exhibiting different degradation kinetics than the shorter isoform, Ret9. In sympathetic neurons, Ret degradation is induced, at least in part, by a complex consisting of the adaptor protein CD2AP and the E3-ligase Cbl-3/c. Knockdown of Cbl-3/c using siRNA reduced the GDNF-induced ubiquitination and degradation of Ret51 in neurons and podocytes, suggesting that Cbl-3/c was a predominant E3 ligase for Ret. Coexpression of CD2AP with Cbl-3/c augmented the ubiquitination of Ret51 as compared with the expression of Cbl-3/c alone. Ret51 ubiquitination by the CD2AP·Cbl-3/c complex required a functional ring finger and TKB domain in Cbl-3/c. The SH3 domains of CD2AP were sufficient to drive the Cbl-3/c-dependent ubiquitination of Ret51, whereas the carboxyl-terminal coiled-coil domain of CD2AP was dispensable. Interestingly, activated Ret induced the degradation of CD2AP, but not Cbl-3/c, suggesting a potential inhibitory feedback mechanism. There were only two major ubiquitination sites in Ret51, Lys¹⁰⁶⁰ and Lys¹¹⁰⁷, and the combined mutation of these lysines almost completely eliminated both the ubiquitination and degradation of Ret51. Ret9 was not ubiquitinated by the CD2AP·Cbl-3/c complex, suggesting that Ret9 was down-regulated by a fundamentally different mechanism. Taken together, these results suggest that only the SH3 domains of CD2AP were necessary to enhance the E3 ligase activity of Cbl-3/c toward Ret51.     

A Conserved NS3 Surface Patch Orchestrates NS2 Protease Stimulation,NS5A Hyperphosphorylation and HCV Genome Replication

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly.     

p66shc Inhibits Insulin-like Growth Factor-I Signaling via Direct Binding to Src through Its Polyproline and Src Homology 2 Domains, Resulting in Impairment of Src Kinase Activation

p66^shc is increased in response to cell stress, and these increases regulate growth factor actions. These studies were conducted to determine how p66^shc alters IGF-I-stimulated Src activation, leading to decreased IGF-I actions. Our results show that p66^shc binds to Src through a polyproline sequence motif contained in the CH2 domain, a unique domain in p66^shc, and IGF-I stimulates this interaction. Disruption of this interaction using a synthetic peptide containing the p66^shc polyproline domain or expression of a p66^shc mutant containing substitutions for the proline residues (P47A/P48A/P50A) resulted in enhanced Src kinase activity, p52^shc phosphorylation, MAPK activation, and cell proliferation in response to IGF-I. To determine the mechanism of inhibition, the full-length CH2 domain and intact p66^shc were tested for their ability to directly inhibit Src kinase activation in vitro. The CH2 domain peptide was clearly inhibitory, but full-length p66^shc had a greater effect. Deletion of the C-terminal Src homology 2 domain in p66^shc reduced its ability to inhibit Src kinase activation. These findings demonstrate that p66^shc utilizes a novel mechanism for modulating Src kinase activation and that this interaction is mediated through both its collagen homologous region 2 and Src homology 2 domains.     

Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein,BCR, and Regulates Levels of Rac1-GTP

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.     

Bradykinin B2 Receptor-Mediated Mitogen-Activated Protein Kinase Activation in COS-7 Cells Requires Dual Signaling via Both Protein Kinase C Pathway and Epidermal Growth Factor Receptor Transactivation

The signaling routes linking G-protein-coupled receptors to mitogen-activated protein kinase (MAPK) may involve tyrosine kinases, phosphoinositide 3-kinase gamma (PI3Kgamma), and protein kinase C (PKC). To characterize the mitogenic pathway of bradykinin (BK), COS-7 cells were transiently cotransfected with the human bradykinin B(2) receptor and hemagglutinin-tagged MAPK. We demonstrate that BK-induced activation of MAPK is mediated via the alpha subunits of a G(q/11) protein. Both activation of Raf-1 and activation of MAPK in response to BK were blocked by inhibitors of PKC as well as of the epidermal growth factor (EGF) receptor. Furthermore, in PKC-depleted COS-7 cells, the effect of BK on MAPK was clearly reduced. Inhibition of PI3-Kgamma or Src kinase failed to diminish MAPK activation by BK. BK-induced translocation and overexpression of PKC isoforms as well as coexpression of inactive or constitutively active mutants of different PKC isozymes provided evidence for a role of the diacylglycerol-sensitive PKCs alpha and epsilon in BK signaling toward MAPK. In addition to PKC activation, BK also induced tyrosine phosphorylation of EGF receptor (transactivation) in COS-7 cells. Inhibition of PKC did not alter BK-induced transactivation, and blockade of EGF receptor did not affect BK-stimulated phosphatidylinositol turnover or BK-induced PKC translocation, suggesting that PKC acts neither upstream nor downstream of the EGF receptor. Comparison of the kinetics of PKC activation and EGF receptor transactivation in response to BK also suggests simultaneous rather than consecutive signaling. We conclude that in COS-7 cells, BK activates MAPK via a permanent dual signaling pathway involving the independent activation of the PKC isoforms alpha and epsilon and transactivation of the EGF receptor. The two branches of this pathway may converge at the level of the Ras-Raf complex.     

Induction of Intrahepatic HCV NS4B,NS5A and NS5B-Specific Cellular Immune Responses following Peripheral Immunization

Numerous studies have suggested that an effective Hepatitis C Virus (HCV) vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.     

Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection

Background

Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated.

Results

Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material.

Conclusion

Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.     

An Autophosphorylation Site of the Protein Kinase SOS2 Is Important for Salt Tolerance in Arabidopsis

The protein kinase SOS2 （Salt Overly Sensitive 2） is essential for salt-stress signaling and tolerance in Arabidopsis. SOS2 is known to be activated by calcium-SOS3 and by phosphorylation at its activation loop. SOS2 is autophosphorylated in vitro, but the autophosphorylation site and its role in salt tolerance are not known. In this study, we identified an autophosphorylation site in SOS2 and analyzed its role in the responses of Arabidopsis to salt stress. Mass spectrometry analysis showed that Ser 228 of SOS2 is autophosphorylated. When this site was mutated to Ala, the autophosphorylation rate of SOS2 decreased. The substrate phosphorylation by the mutated SOS2 was also less than that by the wild-type SOS2. In contrast, changing Ser228 to Asp to mimic the autophosphorylation enhanced substrate phosphorylation by SOS2. Complementation tests in a sos2 mutant showed that the S228A but not the $228D mutation partially disrupted the function of SOS2 in salt tolerance. We also show that activation loop phosphorylation at Thr168 and autophosphorylation at Ser228 cannot substitute for each other, suggesting that both are required for salt tolerance. Our results indicate that Ser 228 of SOS2 is autophosphorylated and that this autophosphorylation is important for SOS2 function under salt stress.     

HCV 5A基因在Hela细胞中的稳定表达及对细胞生长的抑制现象

应用PCR技术从含有HCV(Hepatitis C virus)全长开放阅读框的质粒pBRTM/HCV 1-3011中获得NS5A全长基因片断,利用基因重组技术将其克隆至真核表达载体pcDNA3.1(-)中.通过酶切、PCR及测序鉴定NS5A基因已正确插入到pcDNA3.1(-)中,再利用脂质体介导转染Hela细胞,48h后传代并利用pcDNA3.1(-)质粒上的neo抗性基因加入G-418进行筛选.大约两周后,获得稳定表达的细胞株.经RT-PCR及western blot验证,证实HCV的NS5A基因在Hela细胞中已经获得了表达.在培养条件完全一致的条件下,表达NS5A基因的Hela细胞与pcDNA3.1(-)转染的细胞相比,生长速度明显变慢,其倍增时间约为35-36h,比对照组细胞增加了约50%,而转染pcDNA3.1(-)的细胞的倍增时间与正常Hela细胞则无明显差别,都为23-24h.从而证明HCV的NS5A蛋白具有抑制Hela细胞生长的作用.     

HCV 5A基因在Hela细胞中的稳定表达及对细胞生长的抑制现象

应用PCR技术从含有HCV(Hepatitis Cvirus)全长开放阅读框的质粒pBRTM／HCV 1-3011中获得NS5A全长基因片断,利用基因重组技术将其克隆至真核表达载体pcDNA3．1(-)中。通过酶切、PCR及测序鉴定NS5A基因已正确插入到pcDNA3．1(-)中,再利用脂质体介导转染Hela细胞,48h后传代并利用pcDNA3．1(-)质粒上的neo抗性基因加入G-418进行筛选。大约两周后,获得稳定表达的细胞株。经RT-PCR及western blot验证,证实HCV的NS5A基因在Hela细胞中已经获得了表达。在培养条件完全一致的条件下,表达NS5A基因的Hela细胞与pcDNA3．1(-)转染的细胞相比,生长速度明显变慢,其倍增时间约为35-36h,比对照组细胞增加了约50％,而转染pcDNA3．1(-)的细胞的倍增时间与正常Hela细胞则无明显差别,都为23-24h。从而证明HCV的NS5A蛋白具有抑制Hela细胞生长的作用。     

Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins.

The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates of recombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages. A polyprotein composed of NS2A, NS2B, and the N-terminal 184 amino acids of NS3 was cleaved at the NS2A/NS2B and NS2B/NS3 junctions, whereas a similar polyprotein containing only the first 77 amino acids of NS3 was not cleaved. This finding is consistent with the proposal that the N-terminal 180 amino acids of NS3 constitute a protease domain. Polyproteins containing NS2A and NS3 with large in-frame deletions of NS2B were not cleaved at the NS2A/NS2B or NS2B/NS3 junctions. Coinfection with a recombinant expressing NS2B complemented these NS2B deletions for NS2B/NS3 cleavage and probably also for NS2A/NS2B cleavage. Thus, NS2B is also required for the NS2A/NS2B and NS2B/NS3 cleavages and can act in trans. Other experiments showed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavages in NS3. Indirect evidence that NS3 can also act in trans was obtained. Models are discussed for a two-component protease activity requiring both NS2B and NS3.     

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics,SH3 Binding,and Phosphorylation

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein’s interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191–447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.     

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.     

Expression,Purification, Crystallisation and X-ray Crystallographic Analysis of a Truncated Form of Human Src Homology 2 Containing Inositol 5-Phosphatase 2

The Src homology 2 containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of the phospholipid phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P₃) to form PI(3,4)P₂. PI(3,4,5)P₃ is a key lipid second messenger, which can recruit signalling proteins to the plasma membrane and subsequently initiate numerous downstream signalling pathways responsible for the regulation of a plethora of cellular events such as proliferation, growth, apoptosis and cytoskeletal rearrangements. SHIP2 has been heavily implicated with several serious diseases such as cancer and type 2 diabetes but its regulation remains poorly understood. In order to gain insight into the mechanisms of SHIP2 regulation, a fragment of human SHIP2 containing the phosphatase domain and a region proposed to resemble a C2 domain was crystallized. Currently, no structural information is available on the putative C2-related domain or its relative position with respect to the phosphatase domain. Initial crystals were polycrystalline, but were optimized to obtain diffraction data to a resolution of 2.1 Å. Diffraction data analysis revealed a P2₁2₁2₁ space group with unit cell parameters a = 136.04 Å, b = 175.84 Å, c = 176.89 Å. The Matthews coefficient is 2.54 Å³ Da^?1 corresponding to 8 molecules in the asymmetric unit with a solvent content of 51.7 %.     

2-(3-Thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acids as inhibitors of HCV NS5B RdRp

A series of 2-(3-thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acid inhibitors of the hepatitis C virus (HCV) NS5B polymerase enzyme are reported. Sulfonyl urea substituted analogs in this series proved to be the most potent active site non-nucleoside inhibitors of NS5B reported to date. These compounds had low nanomolar enzyme inhibition across HCV genotypes 1–3 and showed single digit micromolar inhibition in the HCV replicon assay. This improved cell-based activity allowed the binding mode of these compounds to be probed by selection of resistant mutations against compound 21. The results generated are in broad agreement with the previously proposed binding model for this compound class.     

I. Novel HCV NS5B polymerase inhibitors: discovery of indole 2-carboxylic acids with C3-heterocycles

SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC(50)=0.9 μM, replicon EC(50)>100 μM) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC(50)=0.032 μM, replicon EC(50)=1.4 μM) and 7r (NS5B IC(50)=0.017 μM, replicon EC(50)=0.3 μM) with improved enzyme and replicon activity.     

Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-alpha/beta) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein-lacking approximately half of its carboxy-terminal end-showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.