Viral glycoproteins gB and gD of the swine alphaherpesvirus pseudorabies virus (PRV), which is closely related to human herpes simplex virus and varicella-zoster virus, are able to drive internalization of antibodyantigen complexes that may form at the cell surface of infected monocytes, thereby protecting these cells from efficient antibody-mediated lysis. We found earlier that gB relies on an endocytosis motif in its cytoplasmic domain for its function during this internalization process. Here, we report that the PRV gD protein also contains a functional endocytosis motif (YRLL) in its cytoplasmic domain that drives spontaneous endocytosis of gD from the cell surface early in infection and that acts in concert with the endocytosis motif in gB to contribute to efficient internalization of antibody-antigen complexes in PRV-infected monocytes.Alphaherpesviruses have developed numerous strategies to delay or avoid recognition and elimination by different components of the immune system (11,21,45). The swine alphaherpesvirus pseudorabies virus (PRV) especially excels at circumventing antibody-dependent immunity, which allows it to replicate and sometimes spread in pigs that have been vaccinated with an inactivated vaccine (26, 48). PRV-infected blood monocytes play a pivotal role in spread of PRV in the presence of virus-neutralizing antibodies and carry the virus via the blood throughout the body (26).In PRV-infected blood monocytes, like in other PRV-infected cells, newly produced viral envelope proteins are incorporated in the plasma membrane (12,24), thereby rendering the cell recognizable for antibody-dependent immunity (13).However, we found earlier that binding of virus-specific antibodies to viral cell surface proteins in PRV-infected blood monocytes leads to rapid internalization of the antibody-antigen complexes (12), thereby lowering the susceptibility of the infected cell towards antibody-mediated cell lysis (41). This internalization process was found to be clathrin mediated and to depend on two of the PRV proteins at the cell surface, gB and gD (42).Clathrin-mediated endocytosis of cellular transmembrane proteins typically depends on so-called endocytosis motifs in their cytoplasmic domain, most notably YXXL and LL motifs (Y standing for tyrosine, L for leucine, and X for any amino acid). These motifs initiate endocytosis by establishing an interaction with the clathrin-associated AP-2 adaptor complex as a first step in the formation of clathrin-coated vesicles (3,4,20,36).We found that the function of gB in internalization of antibody-antigen complexes from the surface of PRV-infected monocytes depends on a functional tyrosine-based endocytosis motif (YQRL) in its cytoplasmic domain (10), and this motif was found to allow an interaction between gB and the AP-2 complex (43).How PRV gD is involved in internalization of antibodyantigen complexes, on the other hand, is unknown. PRV gD is a type I membrane glycoprotein of 402 amino acids, consisting of an extracellular domain, transmembrane region, and a ...
Mutant strains of pseudorabies virus (PRV) of reduced virulence, such as Bartha or BUK-TK900, have been used for vaccination purposes for many years. In contrast to the Bartha strain, BUK-TK900 has not been well characterised at the molecular level. The detailed analysis of this vaccine strain was urged by the fact of the isolation in Poland of field strains which were suspected to originate from BUK-TK900. We characterised changes in the U(S) region of this strain, focusing our attention on gE and gI genes. The only deletion, about 300 bp, found in BamHI 7 fragment (covering most of the U(S) region) was located in the 28 K (US2) gene. BUK-TK 900 produced small plaques on all cell lines tested in our laboratory (SK6, Vero, MDBK, 3T3). The plaque size was restored to about 70% of wild type virus plaque size when growing BUK-TK900 virus on 3T3 complementing cell line expressing PRV gE and up to 100% when cell line producing gE and gI was used. Both gE and gI genes from BUK-TK900 and from some derivative field isolates have been amplified by PCR reaction but no deletions in these genes have been found. Molecular weight of gene products differed from wild type proteins: gE was bigger than wild type gE while gI was smaller. Both proteins were correctly recognised by all tested polyclonal and monoclonal antibodies. Radioimmunoprecipitation study showed that BUK-TK900 gE and gI interact forming a complex. The whole ORF of BUK-TK900 gE was sequenced and only few point mutations were found; only two of them led to changes of amino acids in the polypeptide chain. These were: methionine at position 124 replaced by threonine and glutamine at position 162 replaced by arginine. The introduction of first of these mutations (Met to Thr) to PRV wild type strain NIA-3 resulted in 22% reduction of plaque size. This result confirms the importance of this domain of gE for its function; it was found previously by others that deletion of amino acids 125 and 126 reduced virulence and neurotropism of PRV. More changes were found in BUK-TK900 gI sequence. Over 80% of these changes were located in the terminal 1/3rd of the sequence. Some of these mutations may have significant effect on the secondary structure of gI glycoprotein. The change of the secondary structure may be responsible for the decrease of gI stability and the observed reduction of gI molecular mass.
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