Role of Receptor-Mediated Endocytosis in the Formation of Vaccinia Virus Extracellular Enveloped Particles

The Tyr-X-X-Leu (YxxL) motif of the vaccinia virus F13L protein was examined for late (L) domain activity. The ability of an F13L deletion virus to form plaques was restored by PCR products containing single alanine substitutions within the motif and a YAAL construct but not by constructs lacking both the Y and L residues. Recombinant viruses possessing alanine substitutions in place of the tyrosine or the leucine residue in the YxxL motif demonstrated small, asymmetrical plaques. RNA interference-dependent depletion of Alix and TSG101 (host proteins involved in L domain-dependent protein trafficking) diminished extracellular enveloped virion production to various degrees, suggesting that the YxxL motif is a genuine L domain.

supporting

confidence: 78%

mentioning

confidence: 99%

The Vaccinia Virus F13L YPPL Motif Is Required for Efficient Release of Extracellular Enveloped Virus

Honeychurch

Yang

Jordan

et al. 2007

“…However, HSV-induced cell fusion is usually limited in vitro and in vivo (17), which may result from regulation mechanisms aimed to prevent enhanced pathogenesis (27). It is generally assumed that the increased cell surface expression of viral fusogenic glycoproteins may increase virus-mediated cell-cell fusion (37). Endocytosis could contribute to counteract this process.…”

Section: Discussionmentioning

confidence: 99%

“…Another proposed role of envelope glycoprotein endocytosis in the replication cycle of viruses consists of the regulation of cell-cell fusion (37,64,76). In the case of HSV, fusion for entry and cell-cell fusion require the functional participation of gB, gD, and gH/gL (reviewed in reference 65).…”

Section: Discussionmentioning

confidence: 99%

Contribution of Endocytic Motifs in the Cytoplasmic Tail of Herpes Simplex Virus Type 1 Glycoprotein B to Virus Replication and Cell-Cell Fusion

Zarate

Cantero-Aguilar

Longo

et al. 2007

The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.Herpes simplex virus type 1 (HSV-1), a member of the herpesviruses family, is a ubiquitous human pathogen mainly responsible for infections of mucocutaneous epithelia that may recur due to the virus latency-reactivation cycle. HSV-1 occasionally spreads to the central nervous system, causing severe encephalitis. HSV-1 envelope glycoprotein B (gB), one of the most conserved glycoproteins among herpesviruses, is a major determinant of virus infectivity, in vitro and in vivo. The essential functions of this type 1 transmembrane protein at various steps of viral replication such as entry, fusion, and cell-cell spread, have been extensively studied and in part assigned to specific domains of the protein (11-13, 16, 24, 27, 32, 33, 43, 53, 59, 79), although a precise picture of the mechanisms by which gB fulfills these functions is still missing. Various studies have also illustrated the role of gB in experimental pathogenesis, in particular in HSV-1 neuroinvasion (27,41,78,83). How glycoproteins might be related to neuroinvasion has not been completely elucidated. However, transneuronal spread might be functionally related to the mechanisms of intracellular assembly and the formation of viral mature particles (25,50,69).It is well established that endocytosis pathways are used by numerous viral glycoproteins in infected cells, in a strategy supposedly aimed to help successful replication and to promote pathogenesis (18,45). Endocytosis of herpesviruses glycoproteins has...

“…Antiserum to a peptide corresponding to the N-terminal 15 amino acids of the L2R open reading frame (ORF) followed by a cysteine residue (MEVITD RLDDIVKQNC) was produced in rabbits (Covance, Denver, PA). The viral proteins were detected with rabbit antisera to A17-N (4), A3 (unpublished data), F13 (19), D13 (38), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Covance, Emeryville, CA).…”

Section: Methodsmentioning

confidence: 99%

Participation of Vaccinia Virus L2 Protein in the Formation of Crescent Membranes and Immature Virions

Maruri-Avidal

Domi

Weisberg

et al. 2011

Self Cite

Morphogenesis of vaccinia virus begins with the appearance of crescent-shaped membrane precursors of immature virions in cytoplasmic factories. During the initial characterization of the product of the L2R reading frame, we discovered that it plays an important role in crescent formation. The L2 protein was expressed early in infection and was associated with the detergent-soluble membrane fraction of mature virions, consistent with two potential membrane-spanning domains. All chordopoxviruses have L2 homologs, suggesting an important function. Indeed, we were unable to isolate an infectious L2R deletion mutant. Consequently, we constructed an inducible mutant with a conditional lethal phenotype. When L2 expression was repressed, proteolytic processing of the major core proteins and the A17 protein, which is an essential component of the immature virion membrane, failed to occur, suggesting an early block in viral morphogenesis. At 8 h after infection in the presence of inducer, immature and mature virions were abundantly seen by electron microscopy. In contrast, those structures were rare in the absence of inducer and were replaced by large, dense aggregates of viroplasm. A minority of these aggregates had short spicule-coated membranes, which resembled the beginnings of crescent formation, at their periphery. These short membrane segments at the edge of the dense viroplasm increased in number at later times, and some immature virions were seen. Although the L2 protein was not detected under nonpermissive conditions, minute amounts could account for stunted and delayed viral membrane formation. These findings suggested that L2 is required for the formation or elongation of crescent membranes.