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UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of tegument, the complex protein layer between the capsid and envelope. UL36p performs multiple functions in the HSV life cycle, including a critical but unknown role in capsid cytoplasmic envelopment. We tested whether UL36p is essential for envelopment because it is required to engage capsids with the cellular ESCRT/Vps4 apparatus. A green fluorescent protein (GFP)-fused form of the dominant negative ATPase Vps4-EQ was used to irreversibly tag ESCRT envelopment sites during infection by UL36p-expressing and UL36-null HSV strains. Using fluorescence microscopy and scanning electron microscopy, we quantitated capsid/Vps4-EQ colocalization and examined the ultrastructure of the corresponding viral assembly intermediates. We found that loss of UL36p resulted in a two-thirds reduction in the efficiency of capsid/Vps4-EQ association but that the remaining UL36p-null capsids were still able to engage the ESCRT envelopment apparatus. It appears that although UL36p helps to couple HSV capsids to the ESCRT pathway, this is likely not the sole reason for its absolute requirement for envelopment. IMPORTANCE Envelopment of the HSV capsid is essential for the assembly of an infectious virion and requires the complex interplay of a large number of viral and cellular proteins. Critical to envelope assembly is the virally encoded protein UL36p, whose function is unknown. Here we test the hypothesis that UL36p is essential for the recruitment of cellular ESCRT complexes, which are also known to be required for envelopment.

UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro . Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsid-membrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCE Membrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to the function of UL36p in capsid envelopment.

The K15P membrane protein of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with multiple cellular signaling pathways and is thought to play key roles in KSHV-associated endothelial cell angiogenesis, regulation of B-cell receptor (BCR) signaling, and the survival, activation, and proliferation of BCR-negative primary effusion lymphoma (PEL) cells. Although full-length K15P is ∼45 kDa, numerous lower-molecular-weight forms of the protein exist as a result of differential splicing and poorly characterized posttranslational processing. K15P has been reported to localize to numerous subcellular organelles in heterologous expression studies, but there are limited data concerning the sorting of K15P in KSHV-infected cells. The relationships between the various molecular weight forms of K15P, their subcellular distribution, and how these may differ in latent and lytic KSHV infections are poorly understood. Here we report that a cDNA encoding a full-length, ∼45-kDa K15P reporter protein is expressed as an ∼23- to 24-kDa species that colocalizes with the trans -Golgi network (TGN) marker TGN46 in KSHV-infected PEL cells. Following lytic reactivation by sodium butyrate, the levels of the ∼23- to 24-kDa protein diminish, and the full-length, ∼45-kDa K15P protein accumulates. This is accompanied by apparent fragmentation of the TGN and redistribution of K15P to a dispersed peripheral location. Similar results were seen when lytic reactivation was stimulated by the KSHV protein replication and transcription activator (RTA) and during spontaneous reactivation. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the latent and lytic phases. IMPORTANCE The K15P protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to play key roles in disease, including KSHV-associated angiogenesis and the survival and growth of primary effusion lymphoma (PEL) cells. The protein exists in multiple molecular weight forms, and its intracellular trafficking is poorly understood. Here we demonstrate that the molecular weight form of a reporter K15P molecule and its intracellular distribution change when KSHV switches from its latent (quiescent) phase to the lytic, infectious state. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the viral latent and lytic stages.

Herpes simplex virus (HSV) and, as reported here, pseudorabies virus (PRV) utilize the ESCRT apparatus to drive cytoplasmic envelopment of their capsids. Here, we demonstrate that blocking ESCRT-mediated envelopment using the dominant-negative inhibitor Vps4A-EQ (Vps4A in which glutamate [E] at position 228 in the ATPase active site is replaced by a glutamine [Q]) reduced the ability of HSV and PRV particles to subsequently traffic along microtubules in vitro . HSV and PRV capsid-associated particles with bound green fluorescent protein (GFP)-labeled Vps4A-EQ were readily detected by fluorescence microscopy in cytoplasmic extracts of infected cells. These Vps4A-EQ-associated capsid-containing particles bound to microtubules in vitro but were unable to traffic along them. Using a PRV strain expressing a fluorescent capsid and a fluorescently tagged form of the envelope protein gD, we found that similar numbers of gD-positive and gD-negative capsid-associated particles accumulated in cytoplasmic extracts under our conditions. Both classes of PRV particle bound to microtubules in vitro with comparable efficiency, and similar results were obtained for HSV using anti-gD immunostaining. The gD-positive and gD-negative PRV capsids were both capable of trafficking along microtubules in vitro ; however, motile gD-positive particles were less numerous and their trafficking was more sensitive to the inhibitory effects of Vps4A-EQ. We discuss our data in the context of microtubule-mediated trafficking of naked and enveloped alphaherpesvirus capsids. IMPORTANCE The alphaherpesviruses include several important human pathogens. These viruses utilize microtubule-mediated transport to travel through the cell cytoplasm; however, the molecular mechanisms of trafficking are not well understood. In this study, we have used a cell-free system to examine the requirements for microtubule trafficking and have attempted to distinguish between the movement of so-called “naked” and membrane-associated cytoplasmic alphaherpesvirus capsids.