The wild-type U L 31, U L 34, and U S 3 proteins localized on nuclear membranes and perinuclear virions; the U S 3 protein was also on cytoplasmic membranes and extranuclear virions. The U L 31 and U L 34 proteins were not detected in extracellular virions. U S 3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the U S 3 protein plays an important, but nonessential, role in the egress pathway.Herpes simplex virus type 1 (HSV-1) virions contain a linear double-stranded DNA genome of approximately 152 kb that is packaged into an icosahedral capsid shell. An amorphous tegument layer surrounds the capsid and is, in turn, surrounded by an envelope composed of a host-derived lipid bilayer studded with viral integral membrane proteins. After the viral genome is replicated and packaged into capsids within the nucleus, assembled nucleocapsids acquire a primary lipid envelope by budding through the inner nuclear membrane (INM) into the space located between the inner and outer leaflets of the nuclear envelope (25,33). Whereas the derivation of the primary envelope from the INM is widely accepted, the route of transit of the nascent virions from the perinuclear space to the extracellular space is more controversial. An overview of the key players in herpesvirus egress and a comparison of the salient features of the two proposed envelopment models have been recently published (8,25).A single-step model of herpesvirus envelopment was proposed for the prototypical alphaherpesvirus HSV-1 (6,18,35,44). This model proposes that enveloped virions move through the endoplasmic reticulum (ER) and the Golgi apparatus in transport vesicles with concomitant modification of primary virion glycoproteins. The single-step envelopment model is supported by the observations that (i) enveloped particles within vesicles can be readily detected by electron microscopy and in fracture label studies (35, 44) and (ii) virion egress and virion-associated glycoprotein processing are both inhibited in cells treated with the ionophore monensin (18). On the other hand, neither of these observations can exclude the alternative deenvelopment-reenvelopment model. Such a model is supported by mounting ultrastructural and biochemical evidence (3,10,13,14,30,37,41,46,50) and has been proposed for HSV-1, other alphaherpesviruses such as varicella-zoster virus (VZV) and pseudorabies virus (PrV), and betaherpesviruses such as human cytomegalovirus. In this model, primary envelopment occurs by budding through the INM but the primary envelope surrounding the perinuclear virion is lost, presumably by fusion with the outer lamellae of the nuclear envelope. In a second step, reenvelopment occurs by wrapping of the nucleocapsid and its associated tegument with a lipid bilayer originating from a membranous cytoplasmic organelle bearing viral glycoprotein...

The herpes simplex virus type 1 (HSV-1) U L 34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U L 31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U L 34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U Herpes simplex virus type 1 (HSV-1) nucleocapsids, like those of all herpesviruses are assembled in the nucleus and acquire a lipid bilayer envelope by budding through the inner nuclear membrane into the perinuclear space (10). Several viral proteins have been implicated in this initial budding event, including the myristylated U L 11 protein, glycoprotein K, which is necessary for envelopment in nondividing cells, and U L 34 protein (2, 21, 37). Of these, only U L 34 protein has been implicated solely in the initial envelopment step, whereas glycoprotein K and U L 11 also play roles in egress through the cytoplasm towards the extracellular space (2, 21, 37).The U L 34 sequence predicts that the protein is oriented as a type II integral membrane protein with an N-terminal cytoplasmic domain of 247 amino acids and a C-terminal transmembrane domain of 22 amino acids (32,35,37). The type II membrane topology of HSV-2 U L 34 protein has recently been addressed (39). This topology predicts that if the transmembrane domain were anchored in the outer nuclear membrane, the bulk of the protein would lie in the cytoplasm, whereas localization in the inner nuclear membrane would place the bulk of the protein within the nucleoplasm.The exact role of U L 34 protein in the envelopment process remains unclear. One possibility is that U L 34 protein interacts directly with capsids and/or tegument components and the nuclear membrane, thereby mediating wrapping of the capsid in the membrane. Alternatively, U L 34 protein may be responsible for recruiting other viral or cellular factors to the site of envelopment. Both hypotheses predict that U L 34 protein should associate with the nuclear envelope. To date, research on the localization of HSV-1 U L 34 protein and its homologues in other herpesviruses has not yielded consistent results. In baculovirus-transduced cells. HSV-1 U L 34 protein is found at the nuclear envelope and in the cytoplasm (46), whereas in HSV-1-infected cells, U L 34 protein is reportedly detectable at the cell surface (35). HSV-2 U L 34 protein has been reported to localize at the endoplasmic reticulum in transfected and in-* Corresponding author. Mailing address:

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BackgroundExosomes are membranous nanovesicles secreted into the extracellular milieu by diverse cell types. Exosomes facilitate intercellular communication, modulate cellular pheno/genotype, and regulate microbial pathogenesis. Although human semen contains exosomes, their role in regulating infection with viruses that are sexually transmitted remains unknown. In this study, we used semen exosomes purified from healthy human donors to evaluate the role of exosomes on the infectivity of different strains of HIV-1 in a variety of cell lines.ResultsWe show that human semen contains a heterologous population of exosomes, enriched in mRNA encoding tetraspanin exosomal markers and various antiviral factors. Semen exosomes are internalized by recipient cells and upon internalization, inhibit replication of a broad array of HIV-1 strains. Remarkably, the anti-HIV-1 activity of semen exosomes is specific to retroviruses because semen exosomes blocked replication of the murine AIDS (mAIDS) virus complex (LP-BM5). However, exosomes from blood had no effect on HIV-1 or LP-BM5 replication. Additionally, semen and blood exosomes had no effect on replication of herpes simplex virus; types 1 and 2 (HSV1 and HSV2). Mechanistic studies indicate that semen exosomes exert a post-entry block on HIV-1 replication by orchestrating deleterious effects on particle-associated reverse transcriptase activity and infectivity.ConclusionsThese illuminating findings i) improve our knowledge of the cargo of semen exosomes, ii) reveal that semen exosomes possess anti-retroviral activity, and iii) suggest that semen exosome-mediated inhibition of HIV-1 replication may provide novel opportunities for the development of new therapeutics for HIV-1.

The herpes simplex virus type 1 (HSV-1) US3 kinase is likely important for primary envelopment of progeny nucleocapsids since it localizes to the nuclear envelope of infected cells and largely determines the phosphorylation state and localization of the necessary primary envelopment factor, the UL34 protein. In HEp-2 cells, the production of infectious US3 null progeny is delayed and decreased relative to that of the parental strain, HSV-1(F). Furthermore, the US3 kinase affects the morphology of primary envelopment such that in its absence, UL34 protein-containing enveloped virions accumulate within membrane-bound vesicles. These vesicles are most often found along the interior periphery of the nucleus and may be derived from the inner nuclear membrane. Since the US3 and UL34 proteins comprise a kinase-substrate pair, a reasonable hypothesis is that the US3 kinase influences these replication parameters by direct phosphorylation of the UL34 protein. For this report, recombinant viruses were constructed to determine the significance of UL34 protein phosphorylation and US3 catalytic activity on UL34 protein localization, single-step growth, and envelopment morphology in both HEp-2 and Vero cells. The data presented suggest that the significance of UL34 phosphorylation is cell type dependent and that efficient viral morphogenesis requires US3-mediated phosphorylation of an infected cell protein other than UL34.All known herpesviruses assemble progeny nucleocapsids within the nucleus of the host cell and therefore must have a mechanism to transport nucleocapsids across the nuclear envelope. Among members of the herpesvirus family, nuclear egress has been most extensively studied with herpes simplex viruses types 1 and 2 (HSV-1 and -2) and pseudorabies virus (PRV). Data indicate that progeny nucleocapsids can exit the nucleus of the host cell through envelopment at the inner nuclear membrane and subsequent deenvelopment at the outer nuclear membrane (13, 41). Both envelopment and deenvelopment appear to be facilitated by a specific set of virusencoded (and probably host-encoded) proteins. Gene deletion studies indicate that the HSV-1 envelopment-deenvelopment process involves the UL34, UL31, UL20, UL11, and US3 proteins, although the specific functions of these proteins are unknown (2-4, 11, 15, 16, 34, 35, 37). The envelopment-deenvelopment machinery may include other virus-encoded proteins, and no host-encoded factors have been identified at the time of this report.The UL34 gene of HSV-1 (and PRV) encodes a phosphoprotein that is primarily localized to the nuclear envelope of infected cells and is a necessary component of an envelopment complex that also includes the UL31 protein (34,35,37). Localization data and sequence analysis suggest that the UL34 protein is anchored within the inner nuclear membrane by a C-terminal hydrophobic domain, leaving the bulk of the protein exposed to the interior of the nucleus (34, 35). There are at least three non-mutually exclusive ways by which UL34 protein could facilitate nuc...