The Measles Virus Nucleocapsid Protein Tail Domain Is Dispensable for Viral Polymerase Recruitment and Activity

Paramyxovirus genomes are ribonucleoprotein (RNP) complexes consisting of nucleoprotein (N)-encapsidated viral RNA. Measles virus (MeV) N features an amino-terminal RNA-binding core and a 125-residue tail domain, of which only the last 75 residues are considered fully mobile on the nucleocapsid surface. A molecular recognition element (MoRE) domain mediates binding of the viral phosphoprotein (P). This P N-tail interaction is considered instrumental for recruiting the polymerase complex to the template. We have engineered MeV N variants with tail truncations progressively eliminating the MoRE domain and upstream tail sections. Confirming previous reports, RNPs with N truncations lacking the carboxyl-terminal 43-residues harboring the MoRE domain cannot serve as polymerase template. Remarkably, further removal of all tail residues predicted to be surface-exposed significantly restores RNP bioactivity. Insertion of structurally dominant tags into the central N-tail section reduces bioactivity, but the negative regulatory effect of exposed N-tail stems is sequence-independent. Bioactive nucleocapsids lacking exposed N-tail sections are unable to sustain virus replication, because of weakened interaction of the advancing polymerase complex with the template. Deletion of the N-MoRE-binding domain in P abrogates polymerase recruitment to standard nucleocapsids, but polymerase activity is partially restored when N-tail truncated RNPs serve as template. Revising central elements of the current replication model, these data reveal that MeV polymerase is capable of productively docking directly to the nucleocapsid core. Dispensable for polymerase recruitment, N-MoRE binding to P-tail stabilizes the advancing polymerase-RNP complex and may rearrange unstructured central tail sections to facilitate polymerase access to the template.     

Phosphorylation of Measles Virus Nucleoprotein Affects Viral Growth by Changing Gene Expression and Genomic RNA Stability

The measles virus (MV) nucleoprotein associates with the viral RNA genome to form the N-RNA complex, providing a template for viral RNA synthesis. In our previous study, major phosphorylation sites of the nucleoprotein were identified as S479 and S510. However, the functions of these phosphorylation sites have not been clarified. In this study, we rescued recombinant MVs (rMVs) whose phosphorylation sites in the nucleoprotein were substituted (rMV-S479A, rMV-S510A, and rMV-S479A/S510A) by reverse genetics and used them in subsequent analyses. In a one-step growth experiment, rMVs showed rapid growth kinetics compared with wild-type MV, although the peak titer of the wild-type MV was the same as or slightly higher than those of the rMVs. Time course analysis of nucleoprotein accumulation also revealed that viral gene expression of rMV was enhanced during the early phase of infection. These findings suggest that nucleoprotein phosphorylation has an important role in controlling viral growth rate through the regulation of viral gene expression. Conversely, multistep growth curves revealed that nucleoprotein-phosphorylation intensity inversely correlated with viral titer at the plateau phase. Additionally, the phosphorylation intensity of the wild-type nucleoprotein in infected cells was significantly reduced through nucleoprotein-phosphoprotein binding. Excessive nucleoprotein-phosphorylation resulted in lower stability against RNase and faster turnover of viral genomic RNA. These results suggest that nucleoprotein-phosphorylation is also involved in viral genomic RNA stability.     

Threonine-74 Is a Key Site for the Activity of Clostridium perfringens Alpha-Toxin

A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [⁶⁵Zn]²⁺ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.     

Brain β-Spectrin Phosphorylation: Phosphate Analysis and Identification of Threonine-347 as a Heparin-Sensitive Protein Kinase Phosphorylation Site

Abstract: Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain β-spectrin yielded a stoichiometry of 3.2 ± 0.18 mol of PO₄/mol of β-spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO₄/mol of spectrin heterodimer, respectively. The ³²P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [³²P]orthophosphate showed phosphorylation of only β-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on β-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated β-spectrin showed that [³²P]phosphate groups were incorporated into both serine (>90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an α-chymotryptic ³²P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338–348 on the β1 repeat of β-spectrin_G (βSPIIa) gene. These data suggest that phosphorylation of Thr³⁴⁷, which is localized on the presumptive synapsin I binding domain of β-spectrin_G, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.     

The Major Site of Phosphorylation within the Rous Sarcoma Virus MA Protein Is Not Required for Replication

About one-third of the MA protein in Rous sarcoma virus (RSV) is phosphorylated. Previous analyses of this fraction have suggested that serine residues 68 and 106 are the major sites of phosphorylation. As a follow-up to that study, we have characterized mutants which have these putative phosphorylation sites changed to alanine, either separately or together. None of the substitutions (S68A, S106A, or S68/106A) had an effect on the budding efficiency or infectivity of the virus. Upon examination of the ³²P-labeled viral proteins, we found that the S68A substitution did not affect phosphorylation in vivo at all. In contrast, the S106A substitution prevented all detectable phosphorylation of MA, suggesting that there is only one major site of phosphorylation in MA. We also found that the RSV MA protein is phosphorylated on tyrosine, but the amount was low and detectable only with large numbers of virions and an antibody specific for phosphotyrosine.     

Agrobacterium tumefaciens Site Attachment as a Necessary Prerequisite for Crown Gall Tumor Formation on Potato Discs

The infectivity of Agrobacterium tumefaciens strain B6 was inhibited about 50% when these bacteria were inoculated on potato discs with equal viable cell counts of a weakly virulent strain of A. tumefaciens (B-48) or autoclaved strains of B6 or B-48. Inhibition by B-48 or autoclaved B6 could still be obtained when these cells were added up to a maximum of 10 minutes after the addition of viable B6. Maximum inhibition occurred when these cells were added 10 minutes prior to the addition of B6. There was no inhibition observed when equal cell counts of B6 were added along with a Gram-positive bacterium or yeast cell, while inhibition was observed when these B6 cells were added simultaneously with other Gram-negative cells. These results suggest that a physical, specific bacterial attachment that occurs within 10 minutes is necessary for tumor formation on potato discs.     

Characterization of a Natural Mutation in an Antigenic Site on the Fusion Protein of Measles Virus That Is Involved in Neutralization

Although measles virus is an antigenically monotypic virus, nucleotide sequence analysis of the hemagglutinin and nucleoprotein genes has permitted the differentiation of a number of genotypes. In contrast, the fusion (F) protein is highly conserved; only three amino acid changes have been reported over a 40-year period. We have isolated a measles virus strain which did not react with an anti-F monoclonal antibody (MAb) which we had previously shown to be directed against a dominant antigenic site. This virus strain, Lys-1, had seven amino acid changes compared with the Edmonston strain. We have shown that a single amino acid at position 73 is responsible for its nonreactivity with the anti-F MAb. With the same MAb, antibody-resistant mutants were prepared from the vaccine strain. A single amino acid change at position 73 (R→W) was observed. The possibility of selecting measles virus variants in vaccinated populations is discussed.     

Reverse Mutational Analysis Reveals Threonine-373 as a Potentially Sufficient Phosphorylation Site for Inactivation of the Retinoblastoma Tumor Suppressor Protein (pRB)

Previous studies in our laboratory have shown that constitutive cyclin E expression can alleviate the requirement for cyclin Δ-CDK activity in the inactivation of the retinoblastoma protein (pRb). RbΔCDK, a mutant construct of pRb with 15 of the 16 CDK phosphorylation sites mutated to alanine represses activation of E2F by mitogen, despite cyclin E over-expression. However, restoration of the four cyclin E-CDK2 phosphorylation sites to RbΔCDK renders this construct sensitive to inactivation by CDK phosphorylation. In the present study, we engage a “reverse mutational analysis” by restoring cyclin E-CDK2 phosphorylation sites to RbΔCDK individually and in combinations in an attempt to discover phosphorylation sites on Rb that are critical for inactivation. Surprisingly, we report that, in both rodent and human cells, restoration of threonine-373 to RbΔCDK, alone or in combination with other phospho-resotrations, results in a loss of the constitutively repressive effect of this construct on E2F activation. Further, induction of endogenous cyclin A protein is blocked by RbΔCDK, but not by mutants of RbΔCDK containing a restored threonine-373. Finally, while S phase entry is blocked by expression of RbΔCDK, restoration of threonine-373 largely attenuates this effect. These findings reveal that phosphorylation of threonine-373 by CDK2-cyclin E represent a potentially crucial event in the inactivation of the pRb protein.     

Function of the Herpes Simplex Virus 1 Small Capsid Protein VP26 Is Regulated by Phosphorylation at a Specific Site

Replacement of the herpes simplex virus 1 small capsid protein VP26 phosphorylation site Thr-111 with alanine reduced viral replication and neurovirulence to levels observed with the VP26 null mutation. This mutation reduced VP26 expression and mislocalized VP26 and its binding partner, the major capsid protein VP5, in the nucleus. VP5 mislocalization was also observed with the VP26 null mutation. Thus, we postulate that phosphorylation of VP26 at Thr-111 regulates VP26 function in vitro and in vivo.     

Moesin Is Not a Receptor for Measles Virus Entry into Mouse Embryonic Stem Cells

The involvement of moesin in measles virus (MV) entry was investigated with moesin-positive and -negative mouse embryonic stem (ES) cells. MV infection of these cells was very ineffective and was independent of moesin expression. Furthermore, when these cells were transfected to express human CD46, a 100-fold increase in syncytium formation was observed with these cells and was independent of the expression of moesin. The only obvious difference between moesin-positive and -negative ES cells was the shape of the syncytia formed. Moesin-negative ES cells expressing or not expressing human CD46 formed separate pieces of fragmented syncytia which were torn apart during spreading, whereas ES cells expressing moesin exhibited typical syncytia. In addition, moesin was not detected on the surface of any murine cells or cell lines that we have tested by a flow cytometric assay with moesin-specific antibodies. These findings indicate that murine moesin is neither a receptor nor a CD46 coreceptor for MV entry into mouse ES cells. Moesin is involved in actin filament-plasma membrane interactions as a cross-linker, and it affects only the spreading and shape of MV-mediated syncytia.     

LIN-9 Phosphorylation on Threonine-96 Is Required for Transcriptional Activation of LIN-9 Target Genes and Promotes Cell Cycle Progression

Cell cycle transitions are governed by the timely expression of cyclins, the activating subunits of Cyclin-dependent kinases (Cdks), which are responsible for the inactivation of the pocket proteins. Overexpression of cyclins promotes cell proliferation and cancer. Therefore, it is important to understand the mechanisms by which cyclins regulate the expression of cell cycle promoting genes including subsequent cyclins. LIN-9 and the pocket proteins p107 and p130 are members of the DREAM complex that in G0 represses cell cycle genes. Interestingly, little is know about the regulation and function of LIN-9 after phosphorylation of p107,p130 by Cyclin D/Cdk4 disassembles the DREAM complex in early G1. In this report, we demonstrate that cyclin E1/Cdk3 phosphorylates LIN-9 on Thr-96. Mutating Thr-96 to alanine inhibits activation of cyclins A2 and B1 promoters, whereas a phosphomimetic Asp mutant strongly activates their promoters and triggers accelerated entry into G2/M phase in 293T cells. Taken together, our data suggest a novel role for cyclin E1 beyond G1/S and into S/G2 phase, most likely by inducing the expression of subsequent cyclins A2 and B1 through LIN-9.     

The Receptor-Binding Site of the Measles Virus Hemagglutinin Protein Itself Constitutes a Conserved Neutralizing Epitope

Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature.