L-Particle Production during Primary Replication of Pseudorabies Virus in the Nasal Mucosa of Swine

Abstract

Different tissue culture cell lines infected with a number of alphaherpesviruses produce, in addition to virions, light particles (L particles). L particles are composed of the envelope and tegument components of the virion but totally lack the proteins of the capsid and the virus genome; therefore, they are noninfectious. In this electron microscopy report, we show that L particles are produced during primary replication of the alphaherpesvirus pseudorabies virus (PRV) in the nasal mucosa of experimentally infected swine, its natural host. Although PRV infected different types of cells of the respiratory and olfactory mucosae, PRV L particles were found to be produced exclusively by epithelial cells and fibroblasts. We observed that formation of noninfectious particles occurred by budding of condensed tegument at the inner nuclear membrane and at membranes of cytoplasmic vesicles, resulting in intracisternal and intravesicular L particles, respectively. Both forms of capsidless particles were clearly distinguishable by the presence of prominent surface projections on the envelope and the higher electron density of the tegument, morphological features which were only observed in intravesicular L particles. Moreover, intravesicular but not intracisternal L particles were found to be released by exocytosis and were also identified extracellularly. Comparative analysis between PRV virion and L-particle morphogenesis indicates that both types of virus particles share a common intracellular pathway of assembly and egress but that they show different production patterns during the replication cycle of PRV.The only purpose of a virus is to perpetuate its genome, and this objective is achieved through parasitization of a host cell and subsequent production of infectious progeny. In the case of alphaherpesviruses, virions released from infected cells are the sole product of virus replication with the capacity to infect new cells and thus to maintain the virus in nature. Alphaherpesvirus virions consist of four distinct morphological components, each of them playing an important role in the infectious process: (i) an external lipid bilayer envelope that contains virus-encoded proteins essential for the attachment and fusion of the virus envelope with the plasma membrane of target cells (11,25,36); (ii) a characteristic layer (known as the tegument) consisting of proteins that are transferred into the cytosol immediately after virus entry and that enhance the ability of virions to initiate the process of infection (7,8,32); and (iii) an icosahedral capsid (consisting of 162 capsomers) that contains and transports (iv) a double-stranded linear DNA molecule up to the nuclear envelope, where the capsid interacts with the nuclear pore complexes to release the virus genome into the nucleus (15, 27, 32). After virus gene expression and DNA replication, progeny nucleocapsids assemble in the nucleus and reach the cytoplasm by envelopment followed by deenvelopment at the nuclear membranes. Recent data indicate that the definitive v...

The Capsid-Associated UL25 Protein of the Alphaherpesvirus Pseudorabies Virus Is Nonessential for Cleavage and Encapsidation of Genomic DNA but Is Required for Nuclear Egress of Capsids

“…This indicates that the UL25 protein is necessary for nuclear egress of capsids but not for encapsidation of viral DNA. In the cytoplasm and on the surface of PrV-⌬UL25F-infected RK13 cells, numerous capsidless tegument-containing particles (L, or light, particles [2,40]) were detectable ( Fig. 5C and D), illustrating that secondary envelopment of tegument proteins in the cytoplasm apparently proceeds undisturbed in the absence of UL25.…”

Identificationof Proteins Associated with Murine Gammaherpesvirus68Virions

“…A small number of heterogeneous particles were also present, including naked capsids, which most likely resulted from the loss of the viral envelope from virions or noninfectious enveloped particles during purification. The existence of two or more kinds of enveloped extracellular particles has been documented for other herpesviruses, including human cytomegalovirus, herpes simplex virus type 1 (HSV-1), and pseudorabies virus (5,29,37).…”

Vesicle formation from the nuclear membrane is induced by coexpression of two conserved herpesvirus proteins

“…L-particles and dense bodies are heterogeneous in size but clearly resemble complete viral particles. So far, formation of perinuclear capsidless enveloped viral particles, i.e., vesicular capsidless structures containing noncapsid viral proteins that originate from invagination of the inner nuclear membrane and are located in the perinuclear cleft, has only rarely been observed (28,29). pUL31 has been hypothesized to represent a component of the primary tegument, and pUL34, a type II transmembrane protein, is considered a component of the primary envelope.…”

“…Even in the absence of virus capsids, secondary envelopment in the cytoplasm proceeds, resulting in the formation of so-called ''light'' (L-) particles (27,28). Capsidless enveloped particles (''primary L-particles'') have also been detected in the perinuclear space in electron microscopic analyses, although they are rare in wild-type virus-infected cells.…”

“…Capsidless enveloped particles (''primary L-particles'') have also been detected in the perinuclear space in electron microscopic analyses, although they are rare in wild-type virus-infected cells. This indicates that nucleocapsids may also be dispensable for primary envelopment and vesicle formation at the inner nuclear membrane (3,28,29). To investigate whether pUL34 and pUL31 are sufficient for vesicle formation from the nuclear envelope, we generated cell lines expressing both proteins of the alphaherpesvirus pseudorabies virus (PrV).…”