The herpes simplex virus type 1 (HSV-1) UL20 protein is an important determinant for virion morphogenesis and virus-induced cell fusion. A precise deletion of the UL20 gene in the HSV-1 KOS strain was constructed without affecting the adjacent UL20.

The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion.

The herpes simplex virus type 1 UL20 protein (UL20p) is an important determinant for cytoplasmic virion morphogenesis and virus-induced cell fusion. To delineate the functional domains of the UL20 protein, we generated a panel of single and multiple (cluster) alanine substitutions as well as UL20p carboxyl-terminal truncations. The UL20 mutant genes could be broadly categorized into four main groups: Group I UL20 mutant genes complemented for both virus production and virus-induced cell fusion; Group II UL20 mutant genes did not complement for either virus-induced cell fusion or infectious virus production; Group III UL20 mutant genes complemented for virus-induced cell fusion to variable extents but exhibited substantially decreased ability to complement UL20-null infectious virus production; Group IV mutant genes complemented for infectious virus production but had variable effects on virus-induced cell fusion; this group included two mutants that efficiently complemented for gBsyn3, but not for gKsyn1, virus-induced cell fusion. In addition, certain recombinant viruses with mutations in either the amino or carboxyl termini of UL20p produced partially syncytial plaques on Vero cells in the absence of any other virally encoded syncytial mutations. These studies indicated that the amino and carboxyl termini of UL20p contained domains that functioned both in infectious virus production and virus-induced cell fusion. Moreover, the data suggested that the UL20p's role in virus-induced cell fusion can be functionally separated from its role in cytoplasmic virion morphogenesis and that certain UL20p domains that function in gB-syn3 virus-induced cell fusion are distinct from those functioning in gKsyn1 virus-induced cell fusion.

Final envelopment of the cytoplasmic herpes simplex virus type 1 (HSV-1) nucleocapsid is thought to occur by budding into trans-Golgi network (TGN)-derived membranes. The highly membrane-associated proteins UL20p and glycoprotein K (gK) are required for cytoplasmic envelopment at the TGN and virion transport from the TGN to extracellular spaces. Furthermore, the UL20 protein is required for intracellular transport and cell surface expression of gK. Independently expressed gK or UL20p via transient expression in Vero cells failed to be transported from the endoplasmic reticulum (ER). Similarly, infection of Vero cells with either gK-null or UL20-null viruses resulted in ER entrapment of UL20p or gK, respectively. In HSV-1 wild-type virus infections and to a lesser extent in transient gK and UL20p coexpression experiments, both gK and UL20p localized to the Golgi apparatus. In wild-type, but not UL20-null, viral infections, gK was readily detected on cell surfaces. In contrast, transiently coexpressed gK and UL20p predominantly localized to the TGN and were not readily detected on cell surfaces. However, TGN-localized gK and UL20p originated from endocytosed gK and UL20p expressed at cell surfaces. Retention of UL20p to the ER through the addition of an ER retention motif forced total ER retention of gK, indicating that transport of gK is absolutely dependent on UL20p transport. In all experiments, gK and UL20p colocalized at intracellular sites, including the ER, Golgi, and TGN. These results are consistent with the hypothesis that gK and UL20p directly interact and that this interaction facilitates their TGN localization, an important prerequisite for cytoplasmic virion envelopment and egress.It is widely accepted that herpesvirus egress entails a twostage envelopment process. Initially, capsids assemble within the nuclei and virions acquire an initial envelope by budding into the perinuclear spaces. Subsequently, these enveloped virions lose their envelope by fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes. Mature virions subsequently traffic to cell surfaces, presumably following the cellular secretory pathway (31,54,65). Although several lines of evidence support this model of TGN assembly and egress (8,30,54,76), the specific viral and cellular mechanisms responsible for the targeted trafficking of viral envelope glycoproteins to intracellular sites of virion envelopment are only recently being elucidated.Herpes simplex viruses (HSVs) specify at least 11 virally encoded glycoproteins, as well as several nonglycosylated membrane-associated proteins, that are pivotal in membrane fusion processes during a productive viral infection. Mutations that cause extensive virus-induced cell-to-cell fusion have been mapped to at least four regions of the viral genome: the UL20 gene (1,50,53), the UL24 gene (38, 64), the UL27 gene ...

Multiple amino acid changes within herpes simplex virus type 1 (HSV-1) gB and gK cause extensive virusinduced cell fusion and the formation of multinucleated cells (syncytia). Early reports established that syncytial mutations in gK could not cause cell-to-cell fusion in the absence of gB. To investigate the interdependence of gB, gK, and UL20p in virus-induced cell fusion and virion de-envelopment from perinuclear spaces as well as to compare the ultrastructural phenotypes of the different mutant viruses in a syngeneic HSV-1 (F) genetic background, gB-null, gK-null, UL20-null, gB/gK double-null, and gB/UL20 double-null viruses were constructed with the HSV-1 (F) bacterial artificial chromosome pYEBac102. The gK/gB double-null virus YEbac⌬gB⌬gK was used to isolate the recombinant viruses gBsyn3⌬gK and gBamb1511⌬gK, which lack the gK gene and carry the gBsyn3 or gBamb1511 syncytial mutation, respectively. Both viruses formed small nonsyncytial plaques on noncomplementing Vero cells and large syncytial plaques on gK-complementing cells, indicating that gK expression was necessary for gBsyn3-and gBamb1511-induced cell fusion. Lack of virus-induced cell fusion was not due to defects in virion egress, since recombinant viruses specifying the gBsyn3 or gKsyn20 mutation in the UL19/UL20 double-null genetic background caused extensive cell fusion on UL20-complementing cells. As expected, the gB-null virus failed to produce infectious virus, but enveloped virion particles egressed efficiently out of infected cells. The gK-null and UL20-null viruses exhibited cytoplasmic defects in virion morphogenesis like those of the corresponding HSV-1 (KOS) mutant viruses. Similarly, the gB/gK double-null and gB/UL20 double-null viruses accumulated capsids in the cytoplasm, indicating that gB, gK, and UL20p do not function redundantly in membrane fusion during virion de-envelopment at the outer nuclear lamellae.

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Egress of herpes simplex virus type 1 (HSV-1) from the nucleus of the infected cell to extracellular spaces involves a number of distinct steps, including primary envelopment by budding into the perinuclear space, de-envelopment into the cytoplasm, cytoplasmic reenvelopment, and translocation of enveloped virions to extracellular spaces. UL20/gK-null viruses are blocked in cytoplasmic virion envelopment and egress, as indicated by an accumulation of unenveloped or partially enveloped capsids in the cytoplasm. Similarly, UL11-null mutants accumulate unenveloped capsids in the cytoplasm. To assess whether UL11 and UL20/gK function independently or synergistically in cytoplasmic envelopment, recombinant viruses having either the UL20 or UL11 gene deleted were generated. In addition, a recombinant virus containing a deletion of both UL20 and UL11 genes was constructed using the HSV-1(F) genome cloned into a bacterial artificial chromosome. Ultrastructural examination of virus-infected cells showed that both UL20-and UL11-null viruses accumulated unenveloped capsids in the cytoplasm. However, the morphology and distribution of the accumulated capsids appeared to be distinct, with the UL11-null virions forming aggregates of capsids having diffuse tegument-derived material and the UL20-null virus producing individual capsids in close juxtaposition to cytoplasmic membranes. The UL20/UL11 double-null virions appeared morphologically similar to the UL20-null viruses. Experiments on the kinetics of viral replication revealed that the UL20/UL11 double-null virus replicated in a manner similar to the UL20-null virus. Additional experiments revealed that transiently expressed UL11 localized to the trans-Golgi network (TGN) independently of either gK or UL20. Furthermore, virus infection with the UL11/UL20 double-null virus did not alter the TGN localization of transiently expressed UL11 or UL20 proteins, indicating that these proteins did not interact. Taken together, these results show that the intracellular transport and TGN localization of UL11 is independent of UL20/gK functions, and that UL20/gK are required and function prior to UL11 protein in virion cytoplasmic envelopment.Herpes simplex virus type 1 (HSV-1) morphogenesis occurs in multiple stages within infected cells. Initially, the virion capsid assembles within the nucleus and the virion acquires an initial envelope by budding into the perinuclear spaces (39). Subsequently, these enveloped virions fuse with the outer nuclear lamellae, leading to the accumulation of unenveloped capsids in the cytoplasm. Within the cytoplasm, a number of additional tegument proteins attach to the capsid and the fully tegumented capsids bud into cytoplasmic vesicles, which mostly likely originate from the trans-Golgi network (TGN). Enveloped virions are ultimately secreted to extracellular spaces through the utilization of cellular vesicular trafficking systems (7,19,20,33,40,43). The process by which the tegumented cytoplasmic capsids bud into TGN-derived vesicles is not well unders...

A new oncolytic and fusogenic herpes simplex virus type 1 (HSV-1) was constructed on the basis of the wildtype HSV-1(F) strain. To provide for safety and tumor selectivity, the virus carried a large deletion including one of the two alpha4, gamma(1)34.5, alpha0 genes and the latency-associated transcript region. The gamma(1)34.5 gene, a major neurovirulence factor, was replaced by a gene cassette constitutively expressing the red fluorescent protein gene. Homologous recombination was used to transfer the fusogenic gBsyn3 mutation to the viral genome to produce the OncSyn virus. OncSyn causes extensive virus-induced cell fusion (syncytia) and replicates to higher titers than the parental Onc and HSV-1(F) strains in breast cancer cells. Biochemical analysis revealed that the OncSyn virus retains a stable genome and expresses all major viral glycoproteins. A xenograft mouse model system using MDA-MB-435S-luc (MM4L) human breast cancer cells constitutively expressing the luciferase gene implanted within the interscapular region of animals was used to test the ability of the virus to inactivate breast tumor cells in vivo. Seventy-two mice bearing MM4L breast cancer xenografts were randomly divided into three groups and given two rounds of three consecutive intratumoral injections of OncSyn, inactivated OncSyn, or phosphate-buffered saline 3 days apart. A single round of virus injections resulted in a drastic reduction of tumor sizes (p