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1.  Effects of Stabilization of the gp41 Cytoplasmic Domain on Fusion Activity and Infectivity of SIVmac239 
AIDS Research and Human Retroviruses  2011;27(11):1213-1222.
We investigated the effects of introducing specific sequences that are predicted to affect trimer stability into the CT domain of the SIV Env protein. Two constructs, 3HBai and 3HBaa, with additional GCN4-related sequences in the CT domain (45 aa) had enhanced infectivity, and differed in their fusion activity and trimer stability. Another construct, 3HBii, exhibited a very stable trimeric structure. Pseudotyped virions containing 3HBii retained infectivity despite the lack of syncytia formation. In contrast, 3HBai and 3HBaa, which caused extensive syncytia formation, had a less stable trimeric structure. We observed an inverse correlation between trimer stability and fusion activity but no correlation between syncytia formation activity and infectivity. Quantitative cell–cell fusion assays, analysis of Env incorporation, measurement of ectodomain conformation by CD4 binding, and CCR5 blocking assays indicated differential effects on fusion activity and infectivity of the viruses with Env CT modifications. Differences in interaction with CD4 were not affected by trimer stability and were not related to fusion activity or infectivity. The results indicate that changes in the stability of the CT domain can have significant effects on functional activities of the Env external domain and can impact viral biological properties.
PMCID: PMC3206742  PMID: 21434848
2.  Enhanced Mucosal Immune Responses to HIV Virus-Like Particles Containing a Membrane-Anchored Adjuvant 
mBio  2011;2(1):e00328-10.
Previously, a modified HIV Env protein with a heterologous membrane anchor was found to be incorporated into HIV virus-like particles (VLPs) at 10-fold-higher levels than those of unmodified Env. To further improve the immunogenicity of such VLPs, membrane-anchored forms of bacterial flagellin (FliC) or a flagellin with a truncated variable region (tFliC) were constructed to be incorporated into the VLPs as adjuvants. HIV-specific immune responses induced by the resulting VLPs were determined in a guinea pig model. The VLPs induce enhanced systemic antibody responses by either systemic or mucosal vaccination and enhanced mucosal immunity by a mucosal immunization route, as demonstrated by high levels of HIV-specific serum IgG and mucosal IgG and IgA. The quality of the antibody responses was also improved, as shown by enhanced neutralization capacity. VLPs incorporating FliC were more effective in inducing systemic responses, while VLPs containing tFliC were more effective in inducing mucosal IgA responses. The IgG titers in sera were found to last for at least 5 months without a significant drop. These results indicate that HIV VLPs incorporating high levels of Env and a molecular adjuvant have excellent potential for further development as a prophylactic HIV vaccine.
A prophylactic vaccine is urgently needed to control the spread of HIV/AIDS. Antigens inducing strong systemic and mucosal immune responses are promising as vaccines for this mucosally transmitted disease. We found that novel HIV virus-like particles (VLPs) presenting a high level of Env in its native membrane-bound form and coincorporating an innate immune-signaling adjuvant in the same particles were effective in inducing enhanced systemic and mucosal immunity. As new HIV vaccine candidates, these VLPs bridge the gaps of the innate and adaptive, as well as systemic and mucosal, immune responses, providing a new approach for HIV vaccine development.
PMCID: PMC3039440  PMID: 21325038
3.  Role of the long cytoplasmic domain of the SIV Env glycoprotein in early and late stages of infection 
Retrovirology  2007;4:94.
The Env glycoproteins of retroviruses play an important role in the initial steps of infection involving the binding to cell surface receptors and entry by membrane fusion. The Env glycoprotein also plays an important role in viral assembly at a late step of infection. Although the Env glycoprotein interacts with viral matrix proteins and cellular proteins associated with lipid rafts, its possible role during the early replication events remains unclear. Truncation of the cytoplasmic tail (CT) of the Env glycoprotein is acquired by SIV in the course of adaptation to human cells, and is known to be a determinant of SIV pathogenicity.
We compared SIV viruses with full length or truncated (T) Env glycoproteins to analyze possible differences in entry and post-entry events, and assembly of virions. We observed that early steps in replication of SIV with full length or T Env were similar in dividing and non-dividing cells. However, the proviral DNA of the pathogenic virus clone SIVmac239 with full length Env was imported to the nucleus about 20-fold more efficiently than proviral DNA of SIVmac239T with T Env, and 100-fold more efficiently than an SIVmac18T variant with a single mutation A239T in the SU subunit and with a truncated cytoplasmic tail (CT). In contrast, proviral DNA of SIVmac18 with a full length CT and with a single mutation A239T in the SU subunit was imported to the nucleus about 50-fold more efficiently than SIVmac18T. SIV particles with full length Env were released from rhesus monkey PBMC, whereas a restriction of release of virus particles was observed from human 293T, CEMx174, HUT78 or macrophages. In contrast, SIV with T Envs were able to overcome the inhibition of release in human HUT78, CEMx174, 293T or growth-arrested CEMx174 cells and macrophages resulting in production of infectious particles. We found that the long CT of the Env glycoprotein was required for association of Env with lipid rafts. An Env mutant C787S which eliminated palmitoylation did not abolish Env incorporation into lipid rafts, but prevented virus assembly.
The results indicate that the long cytoplasmic tail of the SIV Env glycoprotein may govern post-entry replication events and plays a role in the assembly process.
PMCID: PMC2242802  PMID: 18081926
4.  Prevention of poxvirus infection by tetrapyrroles 
Prevention of poxvirus infection is a topic of great current interest. We report inhibition of vaccinia virus in cell culture by porphyrins and phthalocyanines. Most previous work on the inhibition of viruses with tetrapyrroles has involved photodynamic mechanisms. The current study, however, investigates light-independent inhibition activity.
The Western Reserve (WR) and International Health Department-J (IHD-J) strains of vaccinia virus were used. Virucidal and antiviral activities as well as the cytotoxicity of test compounds were determined.
Examples of active compounds include zinc protoporphyrin, copper hematoporphyrin, meso(2,6-dihydroxyphenyl)porphyrin, the sulfonated tetra-1-naphthyl and tetra-1-anthracenylporphyrins, selected sulfonated derivatives of halogenated tetraphenyl porphyrins and the copper chelate of tetrasulfonated phthalocyanine. EC50 values for the most active compounds are as low as 0.05 µg/mL (40 nM). One of the most active compounds was the neutral meso(2,6-dihydroxyphenyl)porphyrin, indicating that the compounds do not have to be negatively charged to be active.
Porphyrins and phthalocyanines have been found to be potent inhibitors of infection by vaccinia virus in cell culture. These tetrapyrroles were found to be active against two different virus strains, and against both enveloped and non-enveloped forms of the virus, indicating that these compounds may be broadly effective in their ability to inhibit poxvirus infection.
PMCID: PMC166128  PMID: 12773208
5.  Inactivation of Human Immunodeficiency Virus Type 1 by Porphyrins 
Antimicrobial Agents and Chemotherapy  2002;46(12):3917-3925.
We have evaluated the anti-human immunodeficiency virus (HIV) activity of a series of natural and synthetic porphyrins to identify compounds that could potentially be used as microbicides to provide a defense against infection by sexually transmitted virus. For assays we used an epithelial HeLa-CD4 cell line with an integrated long terminal repeat-β-galactosidase gene. For structure-activity analysis, we divided the porphyrins tested into three classes: (i) natural porphyrins, (ii) metallo-tetraphenylporphyrin tetrasulfonate (metallo-TPPS4) derivatives, and (iii) sulfonated tetra-arylporphyrin derivatives. None of the natural porphyrins studied reduced infection by more than 80% at a concentration of 5 μg/ml in these assays. Some metal chelates of TPPS4 were more active, and a number of sulfonated tetra-aryl derivatives showed significantly higher activity. Some of the most active compounds were the sulfonated tetranaphthyl porphyrin (TNapPS), sulfonated tetra-anthracenyl porphyrin (TAnthPS), and sulfonated 2,6-difluoro-meso-tetraphenylporphine [TPP(2,6-F2)S] and its copper chelate [TPP(2,6-F2)S,Cu], which reduced infection by 99, 96, 94, and 96%, respectively. Our observations indicate that at least some of these compounds are virucidal, i.e., that they render the virus noninfectious. The active compounds were found to inhibit binding of the HIV type 1 gp120 to CD4 and also to completely inhibit the ability of Env proteins expressed from recombinant vectors to induce cell fusion with receptor-bearing target cells. These results support the conclusion that modified porphyrins exhibit substantial activity against HIV and that their target is the HIV Env protein.
PMCID: PMC132794  PMID: 12435696

Results 1-5 (5)