The rapid onset of massive, systemic viral replication during primary HIV/SIV infection and the immune evasion capabilities of these viruses pose fundamental problems for vaccines that depend upon initial viral replication to stimulate effector T cell expansion and differentiation1–5. We hypothesized that vaccines designed to maintain differentiated “effector memory” T cell (TEM) responses5,6 at viral entry sites might improve efficacy by impairing viral replication at its earliest stage2, and have therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), the prototypical inducer of life-long TEM responses7–9. RhCMV vectors expressing SIV Gag, Rev/Nef/Tat, and Env persistently infected rhesus macaques (RM), regardless of pre-existing RhCMV immunity, and primed and maintained robust SIV-specific, CD4+ and CD8+ TEM responses (characterized by coordinate TNF, IFN-γ and MIP-1β expression, cytotoxic degranulation, and accumulation at extra-lymphoid sites) in the absence of neutralizing antibodies. Compared to control RM, these vaccinated RM showed increased resistance to acquisition of progressive SIVmac239 infection upon repeated, limiting dose, intra-rectal challenge, including four animals that controlled rectal mucosal infection without progressive systemic dissemination. These data suggest a new paradigm for AIDS vaccine development: that vaccines capable of generating and maintaining HIV-specific TEM might decrease the incidence of HIV acquisition after sexual exposure.

Human cytomegalovirus (HCMV) replication in epithelial and endothelial cells appears to be important in virus spread, disease, and persistence. It has been difficult to study infection of these cell types because HCMV laboratory strains (e.g., AD169 and Towne) have lost their ability to infect cultured epithelial and endothelial cells during extensive propagation in fibroblasts. Clinical strains of HCMV (e.g., TR and FIX) possess a cluster of genes (UL128 to UL150) that are largely mutated in laboratory strains, and recent studies have indicated that these genes facilitate replication in epithelial and endothelial cells. The mechanisms by which these genes promote infection of these two cell types are unclear. We derived an HCMV UL128-to-UL150 deletion mutant from strain TR, TR⌬4, and studied early events in HCMV infection of epithelial and endothelial cells, and the role of genes UL128 to UL150. Analysis of wild-type TR indicated that HCMV enters epithelial and endothelial cells by endocytosis followed by low-pH-dependent fusion, which is different from the pH-independent fusion with the plasma membrane observed with human fibroblasts. TR⌬4 displayed a number of defects in early infection processes. Adsorption and entry of TR⌬4 on epithelial cells were poor compared with those of TR, but these defects could be overcome with higher doses of virus and the use of polyethylene glycol (PEG) to promote fusion between virion and cellular membranes. High multiplicity and PEG treatment did not promote infection of endothelial cells by TR⌬4, yet virus particles were internalized. Together, these data indicate that genes UL128 to UL150 are required for HCMV adsorption and penetration of epithelial cells and to promote some early stage of virus replication, subsequent to virus entry, in endothelial cells.

A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in Escherichia coli containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.The role of individual herpesvirus genes in virus replication and the pathogenesis of virus-induced disease have been best understood from studies using viral genetics. The propagation of infectious clones of herpesviruses as bacterial artificial chromosomes (BACs) has revolutionized the experimental manipulation of viral genomes. Importantly, this technology has allowed application of tools of prokaryotic molecular biology to the study of viral genetics, thereby facilitating the genetic analysis of many herpesviruses (4-6, 16, 23, 24, 27). Previously, mutagenic approaches based on homologous recombination in eukaryotic cells were limited to genetic manipulation of only the most rapidly replicating and promiscuous herpesviruses, such as herpes simplex virus (HSV) and pseudorabies virus (PRV) (19). In this study, we have adapted a recombination strategy originally developed for mutagenesis of BACs containing mouse chromosomes to mutagenize the human cytomegalovirus (HCMV) genome (8,10,25,26). This methodology takes advantage of a lambda phage recombination system (RED locus) expressed from a temperature-sensitive promoter that enables the use of linear single-stranded DNA to target genes of interest carried on BACs maintained in Escherichia coli (8,26). We have used this recombination system together with a blue/white lacZ-based selection approach to define the importance of HCMV tegument protein pp28 (UL99) for HCMV replication. The product of the HCMV UL99 open reading frame (ORF) is a 190-amino-acid (aa) phosphorylated tegument protein, which is modified by myristylation (17,18,21). Homologues of pp28 are found in all herpesviruses. Studies with the HSV homologue UL11 have indicated that this protein is also myristylated, traffics in the cytoplasm, and is essential for wild-type levels of virus replication in vitro (2, 3, 11-13). Furthermore, more recent studies of the PRV UL11 * Corresponding aut...

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