The V Protein of Canine Distemper Virus Is Required for Virus Replication in Human Epithelial Cells

Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.     

GPS2 Is Required for the Association of NS5A with VAP-A and Hepatitis C Virus Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of the replication complex associated with various cellular proteins. It has been reported that G protein pathway suppressor 2 (GPS2) is a potential NS5A-binding factor, as identified in a yeast two-hybrid screens of human cDNA library using viral proteins as baits [1]. In this study, we demonstrated the interaction between GPS2 and NS5A in mammalian cells by coimmunoprecipitation analysis and found that both exogenously and endogenously expressed GPS2 interacted with NS5A of genotype 1b and 2a. Mutagenesis study demonstrated that Domain I of NS5A and coiled-coil domain of GPS2 are responsible for the interaction. Knockdown of GPS2 in hepatoma cell lines suppressed the replication of HCV RNA, which can be rescued by the expression of an RNAi-resistant GPS2. Furthermore, overexpression of GPS2 enhanced the association of NS5A with a proviral cellular factor, human vesicle-associated membrane protein-associated protein A (VAP-A), while knockdown of GPS2 disrupted interaction between VAP-A and NS5A. Taken together, our results suggest that GPS2 acts as a bridge between NS5A and VAP-A and is required for efficient HCV replication.     

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.     

Nonstructural C Protein Is Required for Efficient Measles Virus Replication in Human Peripheral Blood Cells

The P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V⁻ was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C⁻ progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C⁻-infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C⁻ was as potent as MV tag or MV V⁻ in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.     

Cholesterol Is Required for Surface Transport of Influenza Virus Hemagglutinin

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide–sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid–cholesterol rafts. By reducing the cholesterol level of living cells by 60–70% with lovastatin and methyl-β-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-β-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.     

A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability

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Bluetongue Virus Nonstructural Protein NS3/NS3a Is Not Essential for Virus Replication

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUG^Met→GCC^Ala mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.     

The Amphipathic Helix of Influenza A Virus M2 Protein Is Required for Filamentous Bud Formation and Scission of Filamentous and Spherical Particles

Influenza virus assembles and buds at the infected-cell plasma membrane. This involves extrusion of the plasma membrane followed by scission of the bud, resulting in severing the nascent virion from its former host. The influenza virus M2 ion channel protein contains in its cytoplasmic tail a membrane-proximal amphipathic helix that facilitates the scission process and is also required for filamentous particle formation. Mutation of five conserved hydrophobic residues to alanines within the amphipathic helix (M2 five-point mutant, or 5PM) reduced scission and also filament formation, whereas single mutations had no apparent phenotype. Here, we show that any two of these five residues mutated together to alanines result in virus debilitated for growth and filament formation in a manner similar to 5PM. Growth kinetics of the M2 mutants are approximately 2 logs lower than the wild-type level, and plaque diameter was significantly reduced. When the 5PM and a representative double mutant (I51A-Y52A) were introduced into A/WSN/33 M2, a strain that produces spherical particles, similar debilitation in viral growth occurred. Electron microscopy showed that with the 5PM and the I51A-Y52A A/Udorn/72 and WSN viruses, scission failed, and emerging virus particles exhibited a “beads-on-a-string” morphology. The major spike glycoprotein hemagglutinin is localized within lipid rafts in virus-infected cells, whereas M2 is associated at the periphery of rafts. Mutant M2s were more widely dispersed, and their abundance at the raft periphery was reduced, suggesting that the M2 amphipathic helix is required for proper localization in the host membrane and that this has implications for budding and scission.     

The Pre-NH2-Terminal Domain of the Herpes Simplex Virus 1 DNA Polymerase Catalytic Subunit Is Required for Efficient Viral Replication

The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH₂-terminal domain (residues 1 to 140) within HSV-1 Pol. Due to the conservation of the pre-NH₂-terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5′-3′ polymerase activity. We identified three pre-NH₂-terminal Pol mutants that exhibited 5′-3′ polymerase activity indistinguishable from that of wild-type Pol in vitro: deletion mutants PolΔN43 and PolΔN52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA₆, in which a conserved motif at residues 44 to 49 was replaced with alanines. We constructed the corresponding pol mutant viruses and found that the polΔN43 mutant displayed replication kinetics similar to those of wild-type virus, while polΔN52 and polA₆ mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. Additionally, both polΔN52 and polA₆ viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH₂-terminal domain is important for viral DNA synthesis and production of infectious virus and indicate a functional role for this domain.     

Contribution of NS1 Effector Domain Dimerization to Influenza A Virus Replication and Virulence

Conserved tryptophan-187 facilitates homodimerization of the influenza A virus NS1 protein effector domain. We generated a mutant influenza virus strain expressing NS1-W187R to destabilize this self-interaction. NS1-W187R protein exhibited lower double-stranded RNA (dsRNA)-binding activity, showed a temporal redistribution during infection, and was minimally compromised for interferon antagonism. The mutant virus replicated similarly to the wild type in vitro, but it was slightly attenuated for replication in mice, causing notably reduced morbidity and mortality. These data suggest biological relevance for the W187-mediated homotypic interaction of NS1.     

质粒介导的核衣壳蛋白siRNA干扰A型流感病毒复制的研究

A型流感病毒(Influenza virus)属于正粘病毒科流感病毒属,可引起鸟类、多种禽类、人类和低等哺乳动物的严重疾病[1],该病毒基因组为负链单股RNA病毒, 分8个节段,容易引起抗原漂移和抗原变异,现有的疫苗和药物不能有效的控制该病毒感染.     

质粒介导的核衣壳蛋白siRNA干扰A型流感病毒复制的研究

A型流感病毒(Influenzavirus)属于正粘病毒科流感病毒属,可引起鸟类、多种禽类、人类和低等哺乳动物的严重疾病[1],该病毒基因组为负链单股RNA病毒,分8个节段,容易引起抗原漂移和抗原变异,现有的疫苗和药物不能有效的控制该病毒感染。RNA干扰是由双链RNA(dsRNA)引发的信使RNA(mRNA)序列特异性消减现象,是一种保守的抗病毒机制,已发现于线虫、植物和哺乳动物中[2]。自然发生的RNAi是由一种dsRNA特异的的核酸内切酶Dicer RDE1引发的,它将dsRNA切割成21～23nt的小分子干扰RNA片断(small interferenceRNA,siRNA),siRNA再与某…     

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.