The mechanism by which herpesviruses acquire their tegument is not yet clear. One model is that outer tegument proteins are recruited by the cytoplasmic tails of viral glycoproteins. In the case of herpes simplex virus tegument protein VP22, interactions with the glycoproteins gE and gD have been shown. We have previously shown that the C-terminal half of VP22 contains the necessary signal for assembly into the virus. Here, we show that during infection VP22 interacts with gE and gM, as well as its tegument partner VP16. However, by using a range of techniques we were unable to demonstrate VP22 binding to gD. By using pulldown assays, we show that while the cytoplasmic tails of both gE and gM interact with VP22, only gE interacts efficiently with the C-terminal packaging domain of VP22. Furthermore, gE but not gM can recruit VP22 to the Golgi/trans-Golgi network region of the cell in the absence of other virus proteins. To examine the role of the gE-VP22 interaction in infection, we constructed a recombinant virus expressing a mutant VP22 protein with a 14-residue deletion that is unable to bind gE (⌬gEbind). Coimmunoprecipitation assays confirmed that this variant of VP22 was unable to complex with gE. Moreover, VP22 was no longer recruited to its characteristic cytoplasmic trafficking complexes but exhibited a diffuse localization. Importantly, packaging of this variant into virions was abrogated. The mutant virus exhibited poor growth in epithelial cells, similar to the defect we have observed for a VP22 knockout virus. These results suggest that deletion of just 14 residues from the VP22 protein is sufficient to inhibit binding to gE and hence recruitment to the viral envelope and assembly into the virus, resulting in a growth phenotype equivalent to that produced by deleting the entire reading frame.The herpesvirus tegument is the virion compartment located between the DNA-containing capsid and the virus envelope (6). Although it is well defined that the viral capsid assembles in the nucleus (37,38) and the viral envelope is acquired from cellular membranes (3, 24), the mechanism of tegument protein acquisition is still to be established. At least 20 virusencoded components are recruited into the herpes simplex virus type 1 (HSV-1) tegument (32), and there is increasing evidence to suggest that subsets of these proteins may be added as assembly progresses along the maturation pathway (28). To ensure efficient incorporation, it is likely that individual tegument proteins are specifically targeted to their cellular site of recruitment. Such targeting could involve interaction with a viral partner, a cellular partner, or both. A clearer understanding of how individual tegument proteins are acquired by newly assembling virions will help to define the herpesvirus assembly pathway.A number of protein-protein interactions between individual tegument proteins (13,40,42), and between tegument proteins and glycoproteins (19,20,22,32), have been described that may provide useful insight into the assembly process. In p...

Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear. Here we have constructed a number of recombinant viruses expressing N-and C-terminal truncations of VP22 and have used them to identify regions of the protein involved in its assembly into the virus structure. Analysis of the packaging of these VP22 variants into extracellular virions revealed that the C terminus of VP22 is absolutely required for this process, with removal of the C-terminal 89 residues abrogating its incorporation. However, while these 89 residues alone were sufficient for specific incorporation of small amounts of VP22 into the tegument, efficient packaging of VP22 to the levels of full-length protein required an additional 52 residues of the protein. Coimmunoprecipitation assays indicated that these 52 residues also contained the interaction domain for VP16. Furthermore, analysis of the subcellular localization of the mutant forms of VP22 revealed that only those truncations that were efficiently assembled formed characteristic cytoplasmic trafficking complexes, suggesting that these complexes may represent the cellular location for VP22 assembly into the virus. Taken together, these results suggest that there are two determinants involved in the packaging of VP22-a C-terminal domain and an internal VP16 interaction domain, both of which are required for the efficient recruitment of VP22 to sites of virus assembly.

In Malawian patients with Kaposi sarcoma (KS) and their relatives, we investigated nucleotide-sequence variation in human herpesvirus-8 (HHV-8) subgenomic DNA, amplified from oral and blood samples by use of polymerase chain reaction. Twenty-four people had amplifiable HHV-8 DNA in >1 sample; 9 (38%) were seropositive for human immunodeficiency virus type 1, 21 (88%) were anti-HHV-8-seropositive, and 7 (29%) had KS. Sequence variation was sought in 3 loci of the HHV-8 genome: the internal repeat domain of open-reading frame (ORF) 73, the KS330 segment of ORF 26, and variable region 1 of ORF K1. Significant intraperson/intersample and intrasample sequence polymorphisms were observed in 14 people (60%). For 3 patients with KS, intraperson genotypic differences, arising from nucleotide sequence variations in ORFs 26 and K1, were found in blood and oral samples. For 2 other patients with KS and for 9 people without KS, intraperson genotypic and subgenotypic differences, originating predominantly from ORF K1, were found in oral samples; for the 2 patients with KS and for 4 individuals without KS, intrasample carriage of distinct ORF K1 sequences also were discernible. Our findings imply HHV-8 superinfection.

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To study transmission patterns of human herpesvirus-8 (HHV-8) (Kaposi's sarcoma-associated herpesvirus) in families in Malawi, nucleotide sequences derived from two hypervariable loci of the HHV-8 genome, the V1 and V2 regions of open reading frame K1 (K1/V1 and K1/V2, respectively), were amplified from blood and mouth rinse samples of 22 patients with treated and untreated Kaposi's sarcoma (KS) and their first-degree relatives (n l 67). In patients with KS, vincristine therapy was significantly associated with non-detectability of circulating, but not oral, K1/V1 DNA. Intra-familial K1/V1 phylogenetic comparisons of eight families were possible. Both identical and non-identical sequences were observed between family members, suggesting transmission of HHV-8 along both intra-and extra-familial transmission routes.

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