Sunil K. Lal

The SARS coronavirus nucleocapsid protein induces actin reorganization and apoptosis in COS-1 cells in the absence of growth factors

In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia, and was subsequently proven to be the causative agent of the disease now referred to as SARS (severe acute respiratory syndrome). The complete genome of the SARS-CoV (SARS coronavirus) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) protein shares little homology with other members of the coronavirus family. In the present paper, we show that SARS-CoV N is capable of inducing apoptosis of COS-1 monkey kidney cells in the absence of growth factors by down-regulating ERK (extracellular-signal-regulated kinase), up-regulating JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and affecting their downstream effectors. SARS-CoV N expression also down-regulated phospho-Akt and Bcl-2 levels, and activated caspases 3 and 7. However, apoptosis was independent of the p53 and Fas signalling pathways. Furthermore, activation of the p38 MAPK pathway was found to induce actin reorganization in cells devoid of growth factors. At the cytoskeletal level, SARS-CoV N down-regulated FAK (focal adhesion kinase) activity and also down-regulated fibronectin expression. This is the first report showing the ability of the N protein of SARS-CoV to induce apoptosis and actin reorganization in mammalian cells under stressed conditions.

The nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks S phase progression in mammalian cells

Deregulation of the cell cycle is a common strategy employed by many DNA and RNA viruses to trap and exploit the host cell machinery toward their own benefit. In many coronaviruses, the nucleocapsid protein (N protein) has been shown to inhibit cell cycle progression although the mechanism behind this is poorly understood. The N protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) bears signature motifs for binding to cyclin and phosphorylation by cyclin-dependent kinase (CDK) and has recently been reported by us to get phosphorylated by the cyclin-CDK complex (Surjit, M., Kumar, R., Mishra, R. N., Reddy, M. K., Chow, V. T., and Lal, S. K. (2005) J. Virol. 79, 11476–11486). In the present study, we prove that the N protein of SARS-CoV can inhibit S phase progression in mammalian cell lines. N protein expression was found to directly inhibit the activity of the cyclin-CDK complex, resulting in hypophosphorylation of retinoblastoma protein with a concomitant down-regulation in E2F1-mediated transactivation. Coexpression of E2F1 under such conditions could restore the expression of S phase genes. Analysis of RXL and CDK phosphorylation mutant N protein identified the mechanism of inhibition of CDK4 and CDK2 activity to be different. Whereas N protein could directly bind to cyclin D and inhibit the activity of CDK4-cyclin D complex; inhibition of CDK2 activity appeared to be achieved in two different ways: indirectly by down-regulation of protein levels of CDK2, cyclin E, and cyclin A and by direct binding of N protein to CDK2-cyclin complex. Down-regulation of E2F1 targets was also observed in SARS-CoV-infected VeroE6 cells. These data suggest that the S phase inhibitory activity of the N protein may have major significance during viral pathogenesis.

The nucleocapsid protein of the SARS coronavirus is capable of self-association through a C-terminal 209 amino acid interaction domain.

Abstract Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The virus is a novel coronavirus isolated from patients exhibiting atypical pneumonia and may have originated from wild animals such as civet cats in southern China. The genome of SARS-CoV is a positive-sense, single-stranded RNA whose sequence is distantly related to all known coronaviruses that infect humans and animals. Like other known coronaviruses, SARS-CoV is an enveloped virus containing three outer structural proteins, namely the membrane (M), envelope (E), and spike (S) proteins. The nucleocapsid (N) protein together with the viral RNA genome presumably form a helical core located within the viral envelope. The SARS-CoV nucleocapsid (N) protein is a 423 amino-acid, predicted phospho-protein of 46kDa that shares little homology with other members of the coronavirus family. A short serine-rich stretch, and a putative bipartite nuclear localization signal are unique to it, thus suggesting its involvement in many important functions during the viral life cycle. In this report we have cloned the N gene of the SARS coronavirus, and studied its property of self-association to form dimers. We expressed the N protein as a fusion protein in the yeast two-hybrid system to demonstrate self-association and confirmed dimerization of the N protein from mammalian cell lysates by coimmunoprecipitation. Furthermore, via deletion analysis, we have shown that the C-terminal 209 amino-acid region constitutes the interaction domain responsible for self-association of the N protein to form dimers.

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein Is Phosphorylated and Localizes in the Cytoplasm by 14-3-3-Mediated Translocation

ABSTRACT The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3θ), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3θ we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.

Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis.

Most pandemic influenza virus strains undergo adaptation or reassortment before they acquire the ability to cause fatal infections in a new host species. The pathologic changes and tissue tropism during virus adaptation are not fully understood. Here we investigated pathologic changes and tissue tropism by serial lung-to-lung passaging of human influenza virus strain A/Aichi/2/68 (H3N2) in a BALB/c mouse model. Enhanced pulmonary lesions and systemic virus infection were observed during adaptation. Late passage 10 (P10) virus caused extra-pulmonary spread with necrotic and inflammatory lesions in the brain, heart, spleen and intestine of infected animals, in contrast to infection with earlier passage viruses which were restricted to lungs. Non-conservative mutations in the hemagglutinin (Gly218Glu) and non-structural 1 (Asp125Gly) proteins were identified in P10 virus which exhibited high virulence. Virus growth kinetics showed enhanced replication ability of P10 virus in different cell lines. P10 virus also exhibited the ability to bind to erythrocytes of different host species. These results demonstrate extra-pulmonary spread of influenza virus during adaptation with enhanced replication ability in a new host. This mouse adaptation model may provide a basis for understanding cross-species adaptability corresponding to increased virulence of the influenza A virus, a phenomenon of relevance to the emergence of future highly pathogenic strains.

The Hepatitis E Virus Open Reading Frame 3 Protein Activates ERK through Binding and Inhibition of the MAPK Phosphatase

The hepatitis E virus causes acute viral hepatitis endemic in much of the developing world and is a serious public health problem. However, due to the lack of an in vitro culture system or a small animal model, its biology and pathogenesis are poorly understood. We have shown earlier that the ORF3 protein (pORF3) of hepatitis E virus activates ERK, a member of the MAPK superfamily. Here we have explored the mechanism of pORF3-mediated ERK activation and demonstrated it to be independent of the Raf/MEK pathway. Using biochemical assays, yeast two-hybrid analysis, and intracellular fluorescence resonance energy transfer we showed that pORF3 binds Pyst1, a prototypic member of the ERK-specific MAPK phosphatase. The binding regions in the two proteins were mapped to the N terminus of pORF3 and a central portion of Pyst1. Expression of pORF3 protected ERK from the inhibitory effects of ectopically expressed Pyst1. This is the first example of a viral protein regulating ERK activation by inhibition of its cognate dual specificity phosphatase.

Cytoplasmic Localization of the ORF2 Protein of Hepatitis E Virus Is Dependent on Its Ability To Undergo Retrotranslocation from the Endoplasmic Reticulum

ABSTRACT Hepatitis E virus (HEV) is a positive-strand RNA virus that is prevalent in much of the developing world. ORF2 is the major capsid protein of HEV. Although ORF2 is an N-linked glycoprotein, it is abundantly located in the cytoplasm in addition to having membrane and surface localization. The mechanism by which ORF2 protein obtains access to the cytoplasm is unknown. In this report, we prove that initially all ORF2 protein is present in the endoplasmic reticulum and a fraction of it becomes retrotranslocated to the cytoplasm. The ability of ORF2 to be retrotranslocated is dependent on its glycosylation status and follows the canonical dislocation pathway. However, in contrast to general substrates of the dislocation pathway, retrotranslocated ORF2 protein is not a substrate of the 26S proteasome complex and is readily detectable in the cytoplasm in the absence of any protease inhibitor, suggesting that the retrotranslocated protein is stable in the cytoplasm. This study thus defines the pathway by which ORF2 obtains access to the cytoplasm.

The ORF2 Protein of Hepatitis E Virus Binds the 5′ Region of Viral RNA

ABSTRACT Hepatitis E virus (HEV) is a major human pathogen in much of the developing world. It is a plus-strand RNA virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, ORF1, ORF2, and ORF3. Of these, ORF2 encodes the major capsid protein of the virus and ORF3 encodes a small protein of unknown function. Using the yeast three-hybrid system and traditional biochemical techniques, we have studied the RNA binding activities of ORF2 and ORF3, two proteins encoded in the 3′ structural part of the genome. Since the genomic RNA from HEV has been postulated to contain secondary structures at the 5′ and 3′ ends, we used these two terminal regions, besides other regions within the genome, in this study. Experiments were designed to test for interactions between the genomic RNA fusion constructs with ORF2 and ORF3 hybrid proteins in a yeast cellular environment. We show here that the ORF2 protein contains RNA binding activity. The ORF2 protein specifically bound the 5′ end of the HEV genome. Deletion analysis of this protein showed that its RNA binding activity was lost when deletions were made beyond the N-terminal 111 amino acids. Finer mapping of the interacting RNA revealed that a 76-nucleotide (nt) region at the 5′ end of the HEV genome was responsible for binding the ORF2 protein. This 76-nt region included the 51-nt HEV sequence, conserved across alphaviruses. Our results support the requirement of this conserved sequence for interaction with ORF2 and also indicate an increase in the strength of the RNA-protein interaction when an additional 44 bases downstream of this 76-nt region were included. Secondary-structure predictions and the location of the ORF2 binding region within the HEV genome indicate that this interaction may play a role in viral encapsidation.

An Antibody against a Novel and Conserved Epitope in the Hemagglutinin 1 Subunit Neutralizes Numerous H5N1 Influenza Viruses

ABSTRACT The spread of the recently emerged, highly pathogenic H5N1 avian influenza virus has raised concern. Preclinical studies suggest that passive immunotherapy could be a new form of treatment for H5N1 virus infection. Here, a neutralizing monoclonal antibody (MAb) against the hemagglutinin (HA) of the influenza A/chicken/Hatay/2004 H5N1 virus, MAb 9F4, was generated and characterized. MAb 9F4 binds both the denatured and native forms of HA. It was shown to recognize the HA proteins of three heterologous strains of H5N1 viruses belonging to clades 1, 2.1, and 2.2, respectively. By use of lentiviral pseudotyped particles carrying HA on the surface, MAb 9F4 was shown to effectively neutralize the homologous strain, Hatay04, and another clade 1 strain, VN04, at a neutralization titer of 8 ng/ml. Furthermore, MAb 9F4 also neutralized two clade 2 viruses at a neutralizing titer of 40 ng/ml. The broad cross-neutralizing activity of MAb 9F4 was confirmed by its ability to neutralize live H5N1 viruses of clade 2.2.2. Epitope-mapping analysis revealed that MAb 9F4 binds a previously uncharacterized epitope below the globular head of the HA1 subunit. Consistently, this epitope is well conserved among the different clades of H5N1 viruses. MAb 9F4 does not block the interaction between HA and its receptor but prevents the pH-mediated conformational change of HA. MAb 9F4 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge of mice. Taken together, our results showed that MAb 9F4 is a neutralizing MAb that binds a novel and well-conserved epitope in the HA1 subunit of H5N1 viruses.

PCNA Interacts with Indian Mung Bean Yellow Mosaic Virus Rep and Downregulates Rep Activity

ABSTRACT Proliferative cell nuclear antigen (PCNA), a conserved plant protein as well as an important replication factor, is induced in response to geminivirus infection in the resting cells of the phloem tissues. The biochemical role of PCNA in rolling circle replication (RCR) of geminivirus DNA has not been explored in detail. The initiation of RCR of the bipartite genome of a geminivirus, Indian mung bean yellow mosaic virus (IMYMV), is mainly controlled by viral protein Rep (or AL1 or AC1). The role of host PCNA in RCR of IMYMV was revealed by studying the physical and functional interactions between recombinant PCNA and recombinant IMYMV Rep. Pea nuclear PCNA as well as recombinant pea PCNA showed binding to recombinant Rep in experiments involving both affinity chromatography and yeast two-hybrid approaches. The contacting amino acid residues of PCNA seemed to be present throughout a wide region of the trimeric protein, while those of Rep appeared to be localized only in the middle part of the protein. The site-specific nicking-closing activity and the ATPase function of IMYMV Rep were impaired by PCNA. These observations lead to interesting speculations about the control of viral RCR and dynamic profiles of protein-protein interactions at the RCR origin of the geminiviruses.