Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein gIV in transfected bovine

The modulation of host protein interactions in response to infection provides both a mechanism for virus-mediated control of host protein functions for the benefit of viral replication, while also serving as a signal for the activation of host immune responses to counteract infection (summarized in and ). In BoHV-1, however, phosphorylation of VP22 is required prior to its incorporation into virions (10). Virus yield reduction assay: Monolayer cultures of MDBK cells were grown in 24-well plates and were infected with BHV-1 at a multiplicity of infection of 0.5 TCID50 per cell. Platinum Taq high-fidelity polymerase (Invitrogen) was used to amplify the targeted cDNA using PCR conditions that included hot-start denaturation at 95°C for 30 s followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 1 min, with a final extension step at 72°C for 7 min. Replication kinetics and invasion characteristics of equine herpesvirus-1 and -3 (EHV-1/-3) in nasal and vaginal mucosae were compared using explants. The control plasmid, pBTM116LaminC, encoding a noncognate protein used in the bait dependency test was created as follows. Anti-VP22 polyclonal antibody was raised in mice immunized with purified bacterially expressed VP22 proteins.

). The high level of UL47 is obvious from the protein profile of Wt BHV-1 virions (Fig. However, all these data were derived from studies using herpes simplex virus type 1 (HSV-1) and may not apply to VP22 homologs of other alphaherpesviruses. On the basis of this evidence, we hypothesized BHV-1 antibody exists in immune milk and suppresses HSV-1 activity. Labeled proteins were immunoprecipitated from cell lysates with anti-gB serum, a polyclonal rabbit serum raised against the carboxy terminus of gB. However, it was of interest to scrutinize the effects of the innate defense against the BHVs either in the presence or in the absence of a functional adaptive immune system. This species also reacted with a monoclonal GFP antibody (Clontech), confirming that it represented a novel GFP-UL47 fusion protein (Fig.

Moreover, comparison of the DNA sequences of different BoHV-1 subtypes is generally accepted to show at least 95% homology [32, 33]. BoHV-1 is composed of a double-stranded DNA surrounded by a nucleocapsid, a tegument, and an envelope (2). Nucleocytoplasmic shuttling of VP8 is sensitive to treatment with a RNA polymerase II inhibitor, actinomycin D (52). A plasmid expressing the 51g mutant protein did not transactivate viral promoter activity as efficiently as wt bICP0. Given are the structural components; the bar represents 100nm. In order to identify the role of BHV-1 gG in the viral infection cycle, a gG minus BHV-1 mutant and its gG-positive revertant were constructed and their growth characteristics in Madin-Darby bovine kidney (MDBK) cells were compared. Copyright © 2016, American Society for Microbiology.

The maturation and egress of WT and mutant BoHV-1 was studied showing a process similar to that reported for other alphaherpesviruses. Attachment of the viral glycoproteins to host receptors mediates endocytosis of the virus into the host cell. Recent studies have shown that at least a portion of the tegument structure has an ordered organization and interacts with the capsid (37, 42, 43, 47,48); however, little is known regarding the acquisition of the viral tegument process (14, 15, 36, 38, 41). We have observed an increase in cell viability of confluent monolayers at low TCDD concentrations. In a next step, bovine respiratory and genital mucosa explants of the same animals were inoculated with several BoHV-1 subtypes. Although the mechanism has yet to be defined, we suggest that PLS and β-glucan inhibited BoHV-1 replication by interfering with the early events of viral penetration. This, together with the observation that mutant VP8 is present in virions, albeit in smaller amounts, suggests that the incorporation of VP8 may occur at two stages.

Polyphenols and flavonoids were the major compounds found in T. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. This correlates to lower amounts of mutant VP8 in virions and suggests for the first time that VP8 may be assembled into the virions at two stages, the latter being dependent on phosphorylation. of BHV-1. Proteins encoded by the three BoHV-1 immediate early genes (bICP0, bICP4, and bICP22) and two late proteins (VP16 and gE) were consistently reduced by GSK650394 during early stages of productive infection. The identity of the open reading frame was verified by expression of the authentic recombinant BHV-1 gIV in bovine cells by using eucaryotic expression vectors pRSDneo (strong, constitutive promoter) and pMSG (weak, dexamethasone-inducible promoter). The mucosal surface of the upper respiratory tract plays a major role in EHV-1 replication and transmission.

UL49 homologs are conserved among alphaherpesviruses. Bovine herpesvirus 1 (BHV-1) is a species-specific virus that does not induce cytotoxicity in normal primary human cells but can infect and kill various human immortalized and transformed cell lines.