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.

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 _L 31 protein fused to glutathione- S -transferase (U _L 31-GST) and U _L 34 protein fused to GST (U _L 34-GST) were demonstrated to specifically recognize the U _L 31 and U _L 34 proteins of approximately 34,000 and 30,000 Da, respectively. The U _L 31 and U _L 34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U _L 34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U _L 31 protein at the nuclear rim required the presence of U _L 34 protein, inasmuch as cells infected with a U _L 34 null mutant virus contained U _L 31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U _L 34 protein exclusively at the nuclear rim required the presence of the U _L 31 gene product, inasmuch as U _L 34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U _L 31 null virus. When transiently expressed in the absence of other viral factors, U _L 31 protein localized diffusely in the nucleoplasm, whereas U _L 34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U _L 31 and U _L 34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U _S 3-encoded protein kinase, previously shown to phosphorylate the U _L 34 gene product, U _L 31 and U _L 34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U _S 3 kinase is required for even distribution of U _L 31 and U _L 34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U _L 31 and U _L 34 deletion mutants, these data strongly suggest that the U _L 31 and U _L 34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.

The herpes simplex virus type 1 U _L 34 gene encodes a protein that is conserved in all human herpesviruses. The association of the U _L 34 protein with membranes in the infected cell and its expression as a gamma-1 gene suggest a role in maturation or egress of the virus particle from the cell. To determine the function of this gene product, we have constructed a recombinant virus that fails to express the U _L 34 protein. This recombinant virus, in which the U _L 34 protein coding sequence has been replaced by green fluorescent protein, forms minute plaques and replicates in single-step growth experiments to titers 3 to 5 log orders of magnitude lower than wild-type or repair viruses. On Vero cells, the deletion virus synthesizes proteins of all kinetic classes in normal amounts. Electron microscopic and biochemical analyses show that morphogenesis of the deletion virus proceeds normally to the point of formation of DNA-containing nuclear capsids, but electron micrographs show no enveloped virus particles in the cytoplasm or at the surface of infected cells, suggesting that the U _L 34 protein is essential for efficient envelopment of capsids.

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

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.

Exosomes 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. We 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. These 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.