Role of thiol/disulfide exchange in newcastle disease virus entry

Newcastle disease virus (NDV) entry into host cells is mediated by the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins. We previously showed that production of free thiols in F protein is required for membrane fusion directed by F protein (S. Jain et al., J. Virol. 81:2328-2339, 2007). In the present study we evaluated the oxidation state of F protein in virions and virus-like particles and its relationship to activation of F protein by HN protein, F protein conformational intermediates, and virus-cell fusion. F protein, in particles, does not have free thiols, but free thiols were produced upon binding of particles to target cells. Free thiols were produced at 16°C in F protein in virions bound to the target cells. They also appeared in different fusion defective mutant F proteins. Free thiols were produced in the presence of mutant HN proteins that are defective in F protein activation but are attachment competent. These results suggest that free thiols appear prior to any of the proposed major conformational changes in F protein which accompany fusion activation. These results also indicate that HN protein binding to its receptor likely facilitates the interaction between F protein and host cell isomerases, leading to reduction of disulfide bonds in F protein. Taken together, these results show that free thiols are produced in F protein at a very early stage during the onset of fusion and that the production of free thiols is required for fusion in addition to activation by HN protein.     

Phospholipids in newcastle disease virus infected cells

Infection of chicken cells with Newcastle Disease Virus modifies phosphatidylserine and phosphatidylcholine systhesis in the host cell. The virion contains cellullar phospholipids synthesized both before and after infection. Relative concentration of various labeled phospholipids in the virus differ from those in the corresponding cells and their surface membranes.Late in infection, fragments of membranes with a distribution of labeled phospholipids similar but not identical to that of the virus can be found in the supernatant of infected cells.The significance of these findings is discussed in relation to origin of viral phospholipids and the intervention of the host cell membrane in the assembly of the viral envelope.     

Radioiodination of the envelope proteins of newcastle disease virus

Lactoperoxidase-catalyzed iodination selectively labels the two glycoproteins (VP1 and VP2) of Newcastle disease virus. The low-molecular-weight, nonglycosylated major viral protein, VP6, was not iodinated in the intact virus but was iodinated in disrupted virions, suggesting a localization on the inner, rather than the outer envelope surface. Studies on the distribution of virion proteins labeled with ¹²⁵I and ³H-isoleucine between detergent-soluble and detergent-insoluble fractions show that the virion proteins VP4, VP5, and VP6 are solubilized to a much lesser extent than are VP1 and VP2.     

Photodynamic inactivation of newcastle disease virus with acridine orange

[This corrects the article on p. 1453 in vol. 15.].     

Photodynamic inactivation of newcastle disease virus with acridine orange

Newcastle disease virus in allantoamnionic fluid was photosensitized with acridine orange, and then photodynamically inactivated. The inactivation rate was directly proportional to light intensity and to dye concentrations up to a level of approximately 100 to 200 μg/ml. Inactivation occurred at approximately an exponential rate for the first 4.5 log₁₀ units of virus, and then continued at a decreasing rate.     

Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication

The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3Dpol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.     

Interacting domains of the HN and F proteins of newcastle disease virus

The activation of most paramyxovirus fusion proteins (F proteins) requires not only cleavage of F(0) to F(1) and F(2) but also coexpression of the homologous attachment protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H protein coexpression strongly suggests that an interaction between HN and F proteins is required for fusion, and studies of chimeric HN proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion proteins containing these HN protein sequences but not to GST or to GST containing HN protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant proteins. These mutations inhibited the fusion promotion activities of the proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN protein binds to the F protein HR2 domain, an interaction important for the fusion promotion activity of the HN protein.     

Detection of avian influenza virus and newcastle disease virus by duplex one step RT PCR

Newcastle disease Virus (NDV), a member of the Paramyxoviridae family, and Influenza virus, from the Orthomyxoviridae family, are two main avian pathogens that cause serious economic problems in poultry farming. NDV strains are classified into three major pathotypes: velogenic, mesogenic, and lentogenic. Avian influenza viruses (AIV) are also divided into: low pathogenic (LPAI) and highly pathogenic (HPAI) strains. Both viruses are enveloped, single stranded, negative-sense RNA viruses which give similar symptoms ranging from sub-clinical infections to severe disease, including loss in egg production, acute respiratory syndrome, and high mortality, depending on their level of pathogenicity. This similarity hinders diagnosis when based solely on clinical and post mortem examination. Most of the currently available molecular detection methods are also pathogenspecific, so that more than one RT-PCR is then required to confirm or exclude the presence of both pathogens. To overcome this disadvantage, we have applied a One Step Duplex RT-PCR method to distinguish between those two pathogens. The main objective of the project was to develop a universal, fast, and inexpensive method which could be used in any veterinary laboratory.