Impairment of Nuclear Pores in Bovine Herpesvirus 1-Infected MDBK Cells

Abstract

Herpesvirus capsids originating in the nucleus overcome the nucleocytoplasmic barrier by budding at the inner nuclear membrane. The fate of the resulting virions is still under debate. The fact that capsids approach Golgi membranes from the cytoplasmic side led to the theory of fusion between the viral envelope and the outer nuclear membrane, resulting in the release of capsids into the cytoplasm. We recently discovered a continuum from the perinuclear space to the Golgi complex implying (i) intracisternal viral transportation from the perinuclear space directly into Golgi cisternae and (ii) the existence of an alternative pathway of capsids from the nucleus to the cytoplasm. Here, we analyzed the nuclear surface by high-resolution microscopy. Confocal microscopy of MDBK cells infected with recombinant bovine herpesvirus 1 expressing green fluorescent protein fused to VP26 (a minor capsid protein) revealed distortions of the nuclear surface in the course of viral multiplication. High-resolution scanning and transmission electron microscopy proved the distortions to be related to enlargement of nuclear pores through which nuclear content including capsids protrudes into the cytoplasm, suggesting that capsids use impaired nuclear pores as gateways to gain access to the cytoplasmic matrix. Close examination of Golgi membranes, rough endoplasmic reticulum, and outer nuclear membrane yielded capsid-membrane interaction of high identity to the budding process at the inner nuclear membrane. These observations signify the ability of capsids to induce budding at any cell membrane, provided the fusion machinery is present and/or budding is not suppressed by viral proteins.Herpesviruses comprise the capsid containing the viral genome, the viral envelope consisting of a lipid bilayer with embedded glycoproteins, and tegument proteins filling the space between capsid and envelope. DNA double strands formed during replication are packed into capsids built of proteins imported from the cytoplasm (32). Capsids are transported to the nuclear periphery. Their pathway through the nucleocytoplasmic barrier and the acquisition of tegument and envelope are yet not fully understood (18). Capsids bud through the inner nuclear membrane into the perinuclear space, concomitantly acquiring an envelope (15) and tegument proteins (43). It is assumed that the envelope derived from budding at the inner nuclear membrane fuses with the outer nuclear membrane, releasing both tegument and capsid into the cytoplasmic matrix (4, 6, 7, 12, 14-16, 20, 42, 46). Capsids are then transported to the trans-Golgi network, where they are wrapped by Golgi membranes leading to an enveloped virion within a transport vacuole. Alternatively, it is speculated that virions escape from the perinuclear space via vesicle formation at the outer nuclear membrane (5,7,11,16,31,37). These vesicles then pass the Golgi complex for final maturation of virions. Contradictory to both the fusion and vesicle formation theory is the fact that fully enveloped virions were found in...

Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis

“…This model of virion maturation and egress was challenged by two alternative routes. The first alternative way involves primary envelopment at the inner nuclear membrane followed by intra-luminal transport through the endoplasmic reticulum and Golgi apparatus secretory pathway [108,169,218]. The second alternative route of egress involves the dilation of nuclear pores resulting in direct access of capsids to the cytoplasm.…”

“…The second alternative route of egress involves the dilation of nuclear pores resulting in direct access of capsids to the cytoplasm. Naked capsids would acquire their envelope by budding at any cell membrane [108,218]. Several electron and confocal microscopic observations favoring this route of egress in BoHV-1 [218] and in HSV-1 [108] infected cells emphasize the need for further study in elucidating alphaherpesvirus egress.…”

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

“…As a member of the Alphaherpesvirinae subfamily, HSV-1 is able to establish latency or productive infection where the virus undergoes entry, viral gene expression, and DNA replication. Upon capsid assembly, viral particles exit the nucleus by budding through the inner nuclear membrane (2)(3)(4). In doing so, HSV-1 triggers conformational changes of the nuclear lamina lining the inner nuclear membrane (5), which favors nuclear egress of nucleocapsids (6).…”

“…Herpes simplex virus type 1 (HSV-1) 4 is a human pathogen responsible for human diseases such as keratitis and encephalitis (1). As a member of the Alphaherpesvirinae subfamily, HSV-1 is able to establish latency or productive infection where the virus undergoes entry, viral gene expression, and DNA replication.…”

Role of tegument proteins in herpesvirus assembly and egress

“…In each stage of virion assembly and egress, tegument proteins are found to play important roles. There are also lines of evidence suggesting direct exit from the nucleus of unenveloped nucleocapsids via enlarged nuclear pores or transport of perinuclear enveloped nucleocapsids through a continuum of outer nuclear, rough ER and Golgi membranes (Wild et al, 2002;Leuzinger et al, 2005;Wild et al, 2005Wild et al, , 2009. In this review, the functional roles of tegument proteins will be discussed in the context of the envelopment-deenvelopment-reenvelopment model of virion assembly.…”