William W. Roth

Genetic characterization of HIV type 1 Nef-induced vesicle secretion.

The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated. Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge. To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

The molecular epidemiology and drug resistance determination of HIV type 1 subtype B infection in Barbados.

To better understand the emergence of HIV-1 variants in Barbados and the association with transmission modes, we analyzed phylogenetic relationships and genetic variability among HIV-1 strains collected in 1996 from 36 antiretroviral therapy-naive patients. Only subtype B variants were present in this sampling, based on analysis of HIV-1 envelope (env) C2V3, protease (PR), and reverse transcriptase (RT) sequences. The genetic diversity of env sequences was broad (13.9%; range, 5.9-24.9%), suggesting multiple introductions of distinct HIV-1 strains to the island. The frequency of subtype B HIV-1 variants with similar env V3 features, including the tetrameric tips, GPGR and GPGK, the threonine deletion at position 23, and the substitution of threonine to arginine at position 22, was comparable in heterosexual, bisexual, and homosexual patients. Analyses of amino acid variations in PR sequences revealed a lack of major drug resistance-conferring mutations and a high (90%) prevalence of secondary mutations at positions 36, 63, 71, and 77. While the occurrence of 361, 63P, and 71T mutations in Barbadian strains was similar to the global prevalence for subtype B variants, the frequency (64%) of the V77I mutation was more than three times that seen worldwide. Only two RT antiretroviral resistance mutations (M41L and T215Y) were observed, both from a single patient. This comprehensive genetic analysis documents a broad diversity within HIV-1 subtype B in Barbados and suggests a lack of association between particular subtype B variants and transmission modes.

Micro RNA in Exosomes from HIV-Infected Macrophages

Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

Phylogenetic Examination of HIV Type 1 Glycoprotein 120-V3 Sequences in Patients from Rural Georgia

399 THE HIV/AIDS EPIDEMIC in the United States has been dynamic both in its geographic distribution and in the demographic profiles of annual AIDS cases and HIV incidence. During the first decade of the HIV-1 epidemic in the United States the majority of AIDS cases were localized within large urban centers, with most new infections due either to homosexual contact or intravenous drug use.1 This was in marked contrast to the HIV-1 epidemic in Southeast Asia and sub-Saharan Africa in which the virus was spread primarily through heterosexual contact.2 In the United States, male-to-male sexual contact (MSM) and intravenous drug use (IVDU) remain the most frequently reported risk factors for HIV-1 transmission. However, a survey of AIDS cases indicates an increase in the number of persons infected through heterosexual exposure. During the period from 1990 to 1995 the number of AIDS cases attributed to heterosexual contact increased at a rate faster than the numbers of those acquired by other routes.3 During this same period, the AIDS epidemic has expanded into rural areas, especially in the southeastern United States. Since 1988, the Southeast has reported greatest number of new AIDS cases of any region of the United States. Many occurring in small towns and rural communities.4 Evidence suggests that the continuing expansion of the AIDS epidemic in the rural, southeastern United States is due in large part to the acquisition of HIV-1 via heterosexual contact with HIV-1-positive individuals who were infected by MSM and/or IVDU.5 In an attempt to describe the HIV-1 epidemic in rural Georgia more fully, we conducted an epidemiological study of HIV1 in patients attending the Infectious Diseases Clinic at the Medical College of Georgia (MCG, Augusta, GA). These patients are drawn from a large area of Georgia and several adjacent counties of South Carolina. The majority are residents of cities and towns of 10,000±50,000 population. We report proviral DNA sequences for the V3 region of the HIV-1 surface protein, gp120, derived from lymphocyte (mononuclear cell) DNA of 27 patients. Individuals who were at least 18 years of age and HIV-1 positive, with T lymphocyte counts of 400/mm whole blood, were recruited for this study. All patients in the cohort were confirmed for HIV-1 infection by Western blot analysis. Diagnosis of AIDS was made according to the revised 1993 Centers for Disease Control (CDC, Atlanta, GA) AIDS classification system. Blood was collected by venipuncture into acid citratedextrose tubes and fractionated by standard Ficoll gradient centrifugation, producing plasma, peripheral blood mononuclear cell (PBMC), and red cell fractions.8 The cells in the PBMC fraction were lysed and DNA was prepared using the reagents in the HCV blood DNA kit (Qiagen, Chatsworth, CA). Patient DNA was amplified by nested polymerase chain reaction (PCR), using a limiting dilution protocol. For each patient sample a series of fourfold dilutions was prepared. Firstround PCR was done with the HIV-1 envelope primers C207 and CO728 using the following thermocycler profile: 95°C for 1 min, 55°C for 1.5 min, 72°C for 2 min; repeated for a total of 40 cycles. A 5-m l aliquot of the first-round PCR products was template for a second-round PCR, using the same profile, with a nested HIV-1 envelope primer pair: sense primer 207NT7 (59 -ATTAATACGACTCACTATAGGG CTGTTAAATGGCAGTCTA-3 9 , which contains the T7 promoter primer [underlined] and nucleotides 7002±7019 of HIV HXB2) and antisense primer V3S (59 ATGTAAAACGACGGCCAGT CAGTAGAAAAATTCCCCT-3 9 , which contains the M13-21 primer [underlined] and the complement to nucleotides 7359±7377 of HIV-1 HXB2).9 The PCR products were gel purified and sequenced directly using M13-21 and T7 promoter primers. Sequencing reactions were performed with the Amplitaq FS dye terminator kit and electrophoresed on a 373A DNA sequencer (ABI-Perkin Elmer, Foster City, CA). For each DNA sample duplicate dilution sets were made. For each set of serial dilutions the least concentrated dilution yielding a PCR product was used for analysis. Most of the duplicate PCRs generated identical PCR products. When the duplicate PCRs produced slightly differing sequences, a third PCR was done, after which the product sequences were aligned and a

Application of random amplified polymorphic DNA PCR for genomic analysis of HIV-1-infected individuals

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) is a DNA fingerprinting technique used to detect genomic polymorphisms. We employed sixteen different RAPD-PCR 10-mer primers to amplify DNA from the peripheral blood mononuclear cells (PBMC) of 80 HIV-1-infected individuals. These individuals were previously identified as either heterozygotes (+ /delta32) and homozygotes (+/+) for the CCR5 locus by PCR with gene specific primers. Four of the sixteen randomly selected RAPD primers produced distinguishable banding profiles between CCR5 (+/delta32) heterozygotes and CCR5 (+/+ ) homozygotes. Direct sequencing of some RAPD-PCR products obtained with one of the four RAPD primers that were tested yielded clearly readable, but limited sequences, which were similar to portions of the previously published sequences for (+/+ ) homozygotes (98% similarity) and (+/delta32) heterozygotes (87% similarity) of the CCR5 alleles. Thus, the RAPD-PCR technique may be useful for the identification of human molecular markers that may correlate with susceptibility to HIV-1-infection, or differences in disease progression among HIV-l-infected individuals.