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Prophylactic vaccination of rhesus macaques with rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) elicits immune responses that stringently control highly pathogenic SIV infection, with subsequent apparent clearance of the infection, in ~50% of vaccinees. In contrast, here, we show that therapeutic RhCMV/SIV vaccination of rhesus macaques previously infected with SIV and given continuous combination antiretroviral therapy (cART) beginning 4–9 d post-SIV infection does not mediate measurable SIV reservoir clearance during over 600 d of follow-up on cART relative to RhCMV/control vaccination. However, none of the six animals started on cART on day four or five, across both RhCMV/SIV- and RhCMV/control-vaccinated groups, those rhesus macaques with SIV reservoirs most closely resembling those of prophylactically RhCMV/SIV-vaccinated and protected animals early in their course, showed post-cART viral rebound with up to nine months of follow-up. Moreover, at necropsy, these rhesus macaques showed little to no evidence of replication-competent SIV. These results suggest that the early SIV reservoir is limited in durability and that effective blockade of viral replication and spread in this critical time window by either pharmacologic or immunologic suppression may result in reduction, and potentially loss, of rebound-competent virus over a period of ~two years.

Complete vaccine-mediated immune control of highly pathogenic Mycobacterium tuberculosisis possible if immune effector responses can intercept the infection at its earliest stages.

Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)

Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)

Recombinant vaccinia virus is a useful and powerful tool for the expression and study of foreign genes. Methods that are currently available for the selection of vaccinia virus recombinants include the restoration of viral plaque-forming phenotype, the replication of viral DNA in the presence of BUdR or mycophenolic acid, and the maturation and propagation of virus under antibiotic selection. Though effective, each of these methods requires several weeks of concerted effort to isolate, purify, and amplify a potential recombinant virus. Here we report the development of a bifunctional enzyme (BiZyme) to simplify and expedite the isolation and purification of vaccinia virus recombinants. This novel selection marker is composed of an inframe fusion between the genes encoding gfpand the neomycin phosphotransferase enzyme (neo). Remarkably, expression of the chimeric gfp-neocassette in the presence of G418 confers both viability and fluorescence to transfected or recombinant virus-infected cells, indicating that both activities are retained within the fusion protein. Therfore, BiZyme was incorporated into a recombination plasmid (pGNR) to enable the concomitant insertion of a foreign gene of interest. Here we demonstrate that this selection/amplification process requires a minimum of 11 days to produce the desired vaccinia virus recombinants. Furthermore, recombinants produced in this fashion have been shown to express both biologically active enzymes and antigenically authentic foreign antigens. In addition to its use in the vaccinia virus vector system, the BiZyme bifunctional selection scheme should be applicable to other eukaryotic and prokaryotic expression systems, simply by coupling it to the appropriate host-specific transcription regulatory signals.

Vaccinia virus encodes at least eight proteins that incorporate label from tritiated palmitic acid when it is added to infected cell cultures. Three of these palmitylproteins are encoded by the A33R, B5R, and F13L open reading frames and migrate by gel electrophoresis with relative molecular masses of 23–28, 42, and 37 kDa, respectively. In this report we provide evidence that the A22R and A36R open reading frames also encode palmitylproteins with apparent molecular masses of 22 and 50–55 kDa, respectively. Furthermore, the hemagglutinin protein (A56R) from the Copenhagen strain is shown to be palmitylated while the hemagglutinin protein from the WR and IHD-J strains is not. A 94-kDa VV palmitylprotein appears to be a multimeric complex composed of the B5R protein and possibly others. All vaccinia-encoded palmitylproteins are present in the membranous fraction of cells and are specific for the trans-Golgi network membrane-enveloped forms of the virus, suggesting that these proteins play a role in the envelopment and egress of virions or the infectivity of released virus.

A novel, retinoic acid-induced gene,GRP1-associated scaffoldprotein (GRASP), was isolated from P19 embryonal carcinoma cells using a subtractive screening strategy. GRASP was found to be highly expressed in brain and exhibited lower levels of expression in lung, heart, embryo, kidney, and ovary. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may be a component of a larger protein complex in the cell. Although GRASP does not harbor a predicted membrane spanning domain(s), the protein was observed to be associated with the plasma membrane of transiently transfected mammalian cells. Yeast two-hybrid screening revealed that GRASP interacted strongly with the General Receptor for Phosphoinositides 1(GRP1), a brefeldin A-insensitive guanine nucleotide exchange factor for the ADP-ribosylation factor family of proteins. GRASP·GRP1 interactions were also demonstrated in vitroand in mammalian cells in which GRASP was shown to enhance GRP1 association with the plasma membrane. Furthermore, GRASP colocalized with endogenous ADP-ribosylation factors at the plasma membrane in transfected cells, suggesting that GRASP may modulate signaling by this family of small GTPases.

Vaccinia virus (VV) encodes a 37-kDa envelope protein (p37) that is palmitylated on cysteine residues 185186 of the 372-amino acid protein. We have previously reported on a loosely conserved consensus motif. Further analysis has identified a conserved consensus sequence, Hydro*AAC(C)A(Hydro* represents a hydrophobic portion of a protein determined by any one of the following: a hydrophobic sequence, a transmembrane domain 1–12 amino acids away from the modification site, or the prior addition of a hydrophobic molecule; C, palmitate acceptor cysteines;A,aliphatic residue) that is responsible for directing palmitylation of certain classes of palmitylproteins. We have analyzed the amino acid site occupancy upstreamdownstream of the palmitate acceptor residues in p37 by site-directed mutagenesistransient expression of mutated proteins in VV-infected cells. The two aliphatic alanines naturally found at positions 183184 of the wild-type p37 allow for efficient palmitylation. In contrast, the replacement of leucine at position 187 with glycine increases palmitylation efficiency. The 10 amino acids immediately upstream of the palmitate acceptor site are absolutely necessary while the downstream 10 amino acids are dispensable. These results together with previous data suggests that the Hydro*AAC(C)Amotif is required for efficient palmitylation of p37.

This corrects the article DOI: 10.1038/nature12519

Highly attenuated pp71-deleted RhCMV/SIV vectors elicit immune responses that stringently protect 59% of vaccinated monkeys from SIV challenge.

Live-attenuated, spread-deficient rhesus CMV vectors retain T cell immunogenicity.