Retroviruses are obligate intracellular parasites that carry the information necessary for replication within their genomes. The three polyproteins, Gag, Pol, and Env, encoded by all retroviruses, function to generate progeny virions inside the host cell. Formation of new viral particles requires detailed instructions contained within the Gag polyprotein. Herein we describe our investigation into assembly of the Mason-Pfizer monkey virus (M-PMV). During retrovirus assembly, the transition from immature to a fully infectious mature particle is associated with the operation of molecular switches that trigger dramatic conformational changes of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid lattice is located in the C-terminus of the capsid (CA) protein in many retroviral Gags. The HIV-1 Gag contains a stretch of five amino acid residues termed the 'clasp motif', important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of the HIV-1 and M-PMV highlighted a spacer-like domain in M-PMV that may provide comparable function. In the present study we report an examination of the role of the clasp motif in the M-PMV life cycle. Our results demonstrate that claps motif mutants display major defects in virion assembly and release, and even larger defects in infectivity. Our data identifies the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient viral infection. The retroviral life cycle, unlike that of any other viral family, leads to the obligate integration of a proviral DNA into the host genome of somatic cells and in some cases even into the germ line. This remarkable feature of the Retroviridae family of viruses accounts for their extraordinary persistence through time and widespread abundance among vertebrate hosts. Because retroviral infection can have serious consequences to the host, there is great selective pressure to evolve strong networks that act to control incoming viruses. In the second study presented here, we report a novel cofactor of an antiviral system, Riplet, which operates to augment HIV-1 restriction by ZAP. The zinc-finger antiviral protein (ZAP) is an interferon-stimulated gene (ISG) with potent intrinsic antiviral activity. ZAP inhibits replication of retroviruses including MLV and HIV-1, as well as alphaviruses, filoviruses, hepatitis B virus, etc. ZAP operates at the post-transcriptional stage, reducing the number of viral transcripts available for translation in the cytoplasm, although additional pathways might be at play. The exact mechanisms by which ZAP restricts viral replication are not fully understood. ZAP lacks enzymatic activity and utilizes other cellular proteins to suppress viral replication. TRIM25 and the nuclease KHNYN have been identified as ZAP cofactors, but its activity may well involve other cellular proteins. Here we identify Riplet, a protein known to play a central role in the activation of the retinoic acid-inducible gene I (RIG-I), as a novel ZAP cofactor that acts to augment ZAP’s antiviral activity. Our data demonstrates that Riplet significantly augments ZAP-mediated restriction of HIV-1. Additionally, we show that Riplet interacts with ZAP via its PRY/SPRY domain and that the ubiquitin ligase activity of Riplet is not required to stimulate ZAP-mediated inhibition. Moreover, we show that Riplet interacts with TRIM25 suggesting that both Riplet and TRIM25 may operate synergistically to augment ZAP-mediated inhibition of HIV-1. The intracellular tropism of viruses is determined by a diverse combination of host proteins that allow infection to proceed efficiently. To achieve successful infection the virus needs the contribution of numerous cellular factors that assist at various steps of the life cycle. Conversely, replication requires resistance to species-specific restriction factors that act to suppress virus infection. The replication of M-PMV has been found to be highly restricted in mouse cell lines. The mechanism underlying the restriction of M-PMV replication in mouse cells has not been characterized. In the third study presented here, we examined this potent post-entry block and performed an unbiased genome-wide CRISPR-Cas9 screen, selecting for knock-out of host factors that relieved the block. Our data identified several candidate genes that encode proteins involved in virus trafficking and innate immune activation.

Thesis (DTech (Chemistry))--Cape Peninsula University of Technology, 2016. / Bimetallic silver-platinum (Ag-Pt) nanoparticles (NPs) were synthesized via simultaneous reduction of varying mole fractions of metal precursors H2PtCl6.6H2O and AgNO3 by sodium citrate. Kinetics rates of were as follows; Ag NPs (0.079 s-1), Ag-Pt NPs 1:1 (0.082 s-1) and Pt NPs (0.006 s-1). The UV visible spectrum of Ag NPs exhibited a characteristic absorption band while Pt NPs and Ag-Pt bimetallic NPs exhibited no absorption peaks. Successful formation of both monometallic and bimetallic NPs was confirmed via transmission electron microscopy (TEM); selected area electron diffraction (SAED) and energy dispersive X-ray (EDX) analysis. TEM images depicted core-shell arrangement in the bimetallic (BM) NP ratios (1:1, 1:3 and 3:1) with an average particle size of 21 nm. The particle size trend where monometallic Ag NPs (60 nm) > Pt NPs (2.5 nm) while in the BM ratios Ag-Pt NPs 1:1 (25 nm) > Ag-Pt NPs 1:3 (20.7 nm). X-ray diffraction (XRD) patterns depicted crystallinity in all the synthesized NPs with confirmation of the face centred cubic structure formation. Transducers were fabricated by drop casting the nanoparticless on the glassy carbon electrode (GCE) and their electrochemical properties studied via cyclic voltammetry (CV). High diffusion coefficient (D) and surface coverage reported were Ag NPs (6.70 cm2 s-1, 54.49 mol cm-2 ) and Ag-Pt NPs 1:1 (0.62 cm2 s-11.85 mol cm-2). Electrochemical band gaps ranged from 1.45 to 1.70 eV while the Tauc’s model band gaps of nanoparticles were found in the range of 2.48 to 3.84 eV. These band gaps were found to be inversely proportional to particle size, which was attributed to the quantum confinement effect. Both optical and electrochemical band gap portrayed similar trend as well as an increase in the BM NP relative to monometallics. These nanoparticles band gaps are within semiconductor range for most materials. The electrochemical behaviour and surface characteristics were studied using 0.1 M PBS solution by scan rates variations for the diffusion coefficient determination of modified electrodes which ranged from 0.62 to 6.10 x 10-5 cm2 s-1. Laviron’s approach for parameters such as apparent charge transfer rate constant, ks, and charge transfer coefficient, α, for electron transfer between NPs and GCE were investigated using CV. The values of electron-transfer coefficients ranged from 0.1 to 0.7 while the charge transfer rate constant values ranged from 0.74 to 31.13 s-1.

HIV-1 completes its life cycle by coopting host proteins. Hundreds of proteins have been identified as potential host factors functioning in viral infection through screens, two of which are MID1IP1 and CCT2. Little is known about MID1IP1, but its localization to microtubules may suggest a cytoskeletal function and a possible role in microtubule transport of HIV-1 viral cores. We use the CRISPR/Cas9 system to create frameshift mutations in MID1IP1 in 293 cells and find that these mutations do not produce effects on HIV-1 transduction in experiments capable of assaying for completion of the life cycle from initial entry into host cells to gene expression. Furthermore, we were unable to find an effect on the staining for markers of microtubule stability using Western blots as a result of the mutations in these cells. CCT2 is a component of the TRiC/CCT protein folding complex whose substrates include actin and tubulin, which also suggests that CCT2 might function in the HIV-1 life cycle in a cytoskeleton-dependent manner. siRNA knockdowns in TE671 cells demonstrate a slight effect on HIV-1 transduction. Our data does not support a role for MID1IP1 in the entry stage of the HIV-1 life cycle, but does suggest CCT2 may be a potential candidate for further study.

No description available.

Thesis (MScMedSc) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: The gag gene of HIV-1 encodes for one of the major structural proteins, which contains several conserved cytotoxic T cell (CTL) epitopes. Gag specific CTL responses are important in controlling viral load during acute infection and asymptomatic stages of the infection. Currently, only one complete South African HIV-1 subtype C gag sequence has been published. The first aim of this study was to characterise the complete gag gene of 15 HIV-1 subtype C isolates, to be used as a set of reference sequences in the design of a South African HIV-1 subtype C vaccine. Fifteen HIV-1 subtype C isolates selected for this study, were isolated during 1998 and 1999 from the HIV-1 positive patients attending the Infectious Disease Clinic at Tygerberg Hospital. The gag gene of these isolates was amplified by PCR, cloned into mammalian expression vectors and sequenced. Restriction digest analyses as well as phylogenetic analyses were performed on the sequencing data. Previously published mutational analyses and CTL epitopes were compared to the predicted amino acid sequences of the gag clones. Sequences of 23 complete gag genes representing the 15 HIV-1 subtype C isolates as well as one complete sequence of an HIV-1 subtype B isolate were compiled. Subtyping by restriction fragment length polymorphism (RFLP) would have correctly identified 14 of the 15 subtype C isolates as subtype C and one as unidentifiable. The subtype B isolate would have also been correctly identified. Phylogenetic analyses showed that our subtype C isolates clustered with reference subtype C strains from various countries, including Botswana, India, Israel, Tanzania and Zambia. Strains from Ethiopia and Brazil formed a separate subtype C cluster. The diversity between our isolates was comparable to the diversity seen between all the HIV-1 subtype C strains. Comparisons of previously published mutational analyses and CTL epitopes to the predicted amino acid sequences of the gag clones, showed conservation in most of the clones throughout the sequence. A second aim was to establish transfection and Western Blot techniques in our laboratory for use in future studies. An in vitro transcription! translation assay was performed on the gag clones and the protein producing clones were used to transfect mammalian cells using electroporation. A Western blot was then used to screen for Gag protein expression in the transfected cell Iysates. The in vitro transcription! translation assay showed that seven of the 23 clones could produce a protein of -55 kDa in size. Four out of the seven of these clones gave a weak expression of a-55 kDa protein after transfection in a mammalian cell line. Since the completion of the experimental work of this study, other cloned HIV-1 genes have successfully been transfected into mammalian cells using the electroporation technique and the proteins produced were screened for by Western blot. To conclude with; the native form of the gag gene does not elicit strong expression of the protein, but studies have shown that expression can be improved by sequence-modification of the gag nucleotide sequence. Due to the conservation of gag, the sequence of any subtype C strain can be used for the development of a Southern African vaccine. / AFRIKAANSE OPSOMMING: Die HIV-1 gag geen kodeer vir een van die hoof strukturele proterene en bevat verskeie sitotoksiese T-limfosiet epitope. Gag spesifieke sellulere immuun respons is belangrik vir die beheer van virale lading tydens akute infeksies en tydens asimptomatiese fases van die infeksie. Tans is slegs een volledige Suid Afrikaanse HIV-1 subtipe C nuklerensuur volgorde gepubliseer. Die eerste doel van hierdie studie was om die volledige gag geen van 15 HIV-1 subtipe C isolate te karakteriseer, om gebruik te word as In stel verwysings nukleiensuur volgordes, vir die ontwerp van In Suid Afrikaanse HIV-1 subtipe C entstof. Die 15 HIV-1 subtipe C isolate wat vir hierdie studie geselekteer is, is tydens 1998 en 1999 ge·lsoleer vanaf HIV-1 positiewe pasiente wat die Infeksiesiekte Kliniek, Tygerberg Hospitaal bygewoon het. Die gag geen van hierdie isolate is geamplifiseer deur PKR, gekloneer in soogdier ekspressie vektore en die nukleiensuur volgorde is bepaal. Die nuklerensuur volgorde is gebruik in restriksie ensiem analises asook filogenetiese analises. Reeds gepubliseerde mutasie analises en limfosiet epitope is met die voorspelde aminosuur volgorde van die gag klone vergelyk. Die nukleiensuur volgordes van die 23 volledige gag gene wat die 15 HIV-1 subtipe C isolate verteenwoordig, asook een volledige nukleiensuur volgorde van een HIV-1 subtipe B isolaat, is saamgestel. Subtipering deur middel van restriksie fragment lengte polimorfisme (RFLP) sou 14 uit die 15 subtipe C isolate korrek qerdentifiseer het, maar sou een nie kon identifiseer nie. RFLP sou ook die subtipe B isolaat korrek qerdentifiseer het. Filogenetiese analises het gewys dat ons subtipe C isolate met die verwysings subtipe C stamme van verskeie lande, insluitend Botswana, lndie, Israel, Tanzania en Zambie groepeer. Stamme van Ethiopie en Brasilie het In aparte subtipe C groep gevorm. Die diversiteit tussen ons isolate was vergelykbaar met die diversiteit tussen al die subtipe C stamme. Vergelykings van gepubliseerde mutasie analises en limfosiet epitope met die voorspelde aminosuur volgorde van die gag klone, het konservasie in meeste van die klone, deur die hele nukleiensuur volgorde, getoon. Die tweede doel was om die metodes van transfeksie en Westerse klad in ons laboratorium tot stand te bring. In vitro transkripsie/ translasie toetse is gedoen op die gag klone en die proteten produserende klone is gebruik om soogdierselle te transfekteer deur gebruik te maak van elektroporasie. In Westerse klad is toe gebruik om vir Gag proterenuitdrukkinq in die sellisate te toets. Die in vitro transkripsie/ translasie toets het getoon dat sewe uit 23 klone, In proteren van -55 kDa kon produseer. Vier uit die sewe van hierdie klone het In -55 kDa proteren swak uitgedruk na transfektering van soogdier selle. Sedert die voltooiing van die eksperimentele werk van hierdie stud ie, is ander gekloneerde HIV-1 gene suksesvol in soogdierselle getransfekteer met die gebruik van elektroporasie en die proterene is met In Westerse klad aangetoon. Ten slotte: die natuurlike vorm van die gag geen ontlok nie In sterk ekspressie van die proteren nie, maar ander studies het wei aangetoon dat die ekspressie verbeter kan word met modifikasie van die gag nukleiensuur volgorde. As gevolg van die konservasie van gag, kan die nuklerensuur volgorde van enige subtipe C stam gebruik word vir die ontwikkeling van In Suider Afrikaanse entstof. / The Poliomyelitis Research Foundation / The South African AIDS Vaccine Initiative / Harry Crossley Foundation

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: At the end of 2002, human immunodeficiency virus (HIV) had infected 42 million people worldwide. The morbidity and mortality rate, as well as the epidemic proportions of the disease have led to concentrated scientific efforts to reveal the disease's pathogenesis and develop effective preventative and treatment measures. Advances have been made to inhibit viral replication by suppressing the virus' ability to replicate by developing antiretroviral treatments, although development of a save and effective vaccine is the only way to stem the pandemic. Advances in vaccine design, animal models and clinical research have led to the creation of promising candidate vaccines to counter this rampage, but most of these vaccines entering phase I-III clinical trials are based mainly only subtype B genomes. HIV-1 subtype C is the most commonly transmitted subtype worldwide, and is the predominant subtype in India, China, East and Southern Africa. A subtype C vaccine is critical for the developing nations such as South Africa, where antiretroviral therapies are largely unaffordable. The envelope gene (env) is an attractive target as immunogen to be included in a HIV vaccine. The envelope protein (Env) elicits neutralising antibodies and cytotoxic T-Iymphocyte (CTl) responses. This protein will therefore be useful in creating a humoral and cellular immune response in the host. A shortage in characterised subtype C env gene sequences from South Africa was recognised, and this study focussed on the characterisation of generated sequences, as well as the expression of selected env genes. These immunogens were created for possible use in a prime-boost vaccine modality. The env genes from recent circulating strains in South Africa were amplified by polymerase chain reaction (PCR). The genes were then cloned for sequencing and expression purposes. Phylogenetic relationships were determined by comparing the sequences to reference subtype strains and subtype C strains. Expression of the genes was assessed by Western Blot in 293 cells with HIV- 1 positive patient sera. Sequence analysis showed a more conserved third variable (V3) loop in South African subtype C sequences, with a more variable region downstream from the loop. The crown sequence (GPGQ) and positions of uncharged or negatively charged residues in the V3 loop indicated a non-syncytium-inducing (NSI) phenotype for the isolates. Phylogenetic analysis showed the sequences to all belong to the C subtype, and further that the sequences were not recombinant, which was confirmed by recombination analysis. The intersample diversity observed for strains from South Africa was significantly higher than distances observed to the subtype C consensus sequence. The South African sequences were distributed across several subclusters in a subtype C phylogenetic tree, highlighting the concept that these infections represent a more longstanding epidemic with multiple introductions from different geographic areas. Western Blot with HIV-1 positive patient sera showed the expression of uncleaved gp160 Env proteins, which were Rev dependent. This study has generated much needed subtype C South African env gene sequences that can be used as basis for modification for use as immunogens in a South African vaccine. / AFRIKAANSE OPSOMMING: Teen die einde van 2002 was 42 miljoen mense wêreldwyd geïnfekteer met die menslike immuniteitsgebrekvirus (MIV). Die dode- en sterfte syfers, asook die skaal van die epidemie, het gelei tot 'n wetenskaplike poging om die siekte se patogenese te openbaar en om effektiewe voorkomende en terapeutiese middels te ontwikkel. Vordering is reeds gemaak om die virus se replikasie te hinder deur die ontwerp van antivirale middels, alhoewel die ontwikkeling van 'n doeltreffende en veilige entstof die enigste manier is om die pandemie te stuit. As gevolg van die vordering in entstof ontwerp, diere modelle en kliniese navorsing is belowende kandidaat entstowwe wat die infeksie kan teenwerk ontwikkel, maar die meeste van hierdie enstowwe wat vir fase I-III kliniese proewe gebruik word is gebaseer op subtipe B genome. MIV-subtipe C is wêreldwide die algemeenste subtipe wat oorgedra word en is die oorheersende subtipe in lande soos Indië, China, oostelike en suidelike Afrika. 'n Subtipe C entstof word dringend benodig in ontwikkelende lande soos Suid-Afrika waar antivirale middels onbekostigbaar is. Die membraangeen is 'n aanloklike teiken om as immunogeen in 'n MIV entstof te dien. Die membraanproteïen lok neutraliserende teenliggame en sitotoksiese T-limfosiet reaksies uit. Die proteïen sal dus 'n humorale en sellulêre immuunrespons in die gasheer ontlok. 'n Tekort aan gekarakteriseerde subtipe C membraangeen volgordes van Suid-Afrika is opgemerk, en dus fokus hierdie studie op die karakterisering van gegenereerde volgordes, asook die uitdrukking van geselekteerde membraangene. Die immunogene is geskep om moontlik gebruik te word in 'n stimuleer-versterkingsenstof toedieningstrategie. Die membraangene van onlangs sirkulerende virusstamme in Suid-Afrika was geamplifiseer deur polimerase kettingreaksie (PKR). Die gene is daarna gekloneer vir beide volgordebepalings en uitdrukkingdoeleindes. Filogenetiese verhoudings is uitgewerk deur die volgordes met verwysingsstamme en subtipe C stamme te vergelyk. Uitdrukking van die gene is waargeneem in 293 selle deur die Westerse kladtegniek te gebruik met MIV-1 positiewe pasiëntsera as teenliggaam. Volgorde-analise het aangetoon dat die derde varieerbare (V3) lus meer gekonserveer is, en dat die gedeelte wat op die lus volg meer varieerbaar is. Die kroonvolgorde (GPGQ) asook posisies van ongelaaide of negatief gelaaide aminosure in die V3 lus het aangedui dat die isolate 'n nie-syncytia induserende fenotipe het. Filogenetiese analise het aangedui dat al die volgordes subtipe C is en dat die volgordes nie rekombinant is nie. Dit is ook deur rekombinasie analise bewys. Die inter-monster diversiteit van die Suid-Afrikaanse volgordes was hoër as die waargenome afstand vanaf die subtipe C konsensus volgorde. Die Suid-Afrikaanse volgordes is versprei oor verskeie subgroepe in 'n subtipe C boom, wat die konsep dat hierdie infeksies 'n meer gevestigde epidemie voorstel waar veelvuldige infeksies met verskillende geografiese oorspronge plaasgevind het beklemtoon. Die Westerse klad het ongeprosesseerde gp160 membraanproteïne aangetoon wat Rev afhanklik was. Hierdie studie het hoogs benodigde subtipe C Suid-Afrikaanse volgordes van membraangene geproduseer. Die volgordes kan as basis dien om die gene te modifiseer sodat dit gebruik kan word as immunogene in 'n entstof vir Suid-Afrika.

No description available.

The researcher is propelled and motivated by the Minister of Public Service Association, Geraldine Frasser-Moleketi, recognising the serious nature of HIV and AIDS and its impact on South Africa when she initiated the Impact and Action Project in January 2000, which was aimed at ensuring that Public Service is able to sustain a quality service in spite of pandemic: AIDS. The apprehension of writing on the theme is to enlighten both the employer and employees with their obligations, rights and legal framework in the management of the pandemic: AIDS within the employment perspective. The eminence of the study is based on the participation of management and their intervention, because without their involvement, AIDS deaths translate into lost productivity, increased high rate of absenteeism, short or long illnesses, and lower morale of employees and hampering of rendering of services. The study comprises of five chapters, containing the orientation of the study; literature review; research design and methodology; data analysis and results and summary, findings and recommendation, respectively. The instrument employed to gather data was applied in five regions, and data is gathered from a target sample of 96 out of 2084 employees. Please note in order to avoid the continuous use of "HN and AIDS", virus, epidemic, disease or cumbersome descriptions, the noun pandemic: AIDS, will be used and will denote "HIV and AIDS". / MBA (HRM) North-West University, Mafikeng Campus, 2005

Despite significant medical advances in HIV treatment, people living with HIV and substance use (PLWH-SU) remain left behind. Compared to people living with HIV (PLWH) without comorbid substance use, PLWH-SU are less likely to engage in medical care and to achieve viral suppression. As a result, PLWH-SU have more frequent preventable hospitalizations, higher rates of viral transmission, and greater morbidity and mortality. Although there is extensive research that explores ways to enhance PLWH-SU’s engagement in HIV care by improving patient-provider interactions, most have focused on the patient, and none have been effective. Grounded in the sociological theory of habitus, this dissertation attended to the medical provider in the patient-provider dyad and aimed to better understand how medical providers’ perceptions and dispositions towards PLWH-SU are formed, and how these perceptions and dispositions are displayed in the ways medical providers interact with and take care of PLWH-SU. Before engaging with habitus, I first conducted a systematic review on how the theory has been used to study medical providers’ clinical practices. Results of the review show that while existing literature has been limited and unclear in its usage of habitus, these studies are informative, and they demonstrate that habitus can be a suitable theoretical foundation for expanding present approaches to research on medical providers’ clinical interactions with PLWH-SU. Following the systematic review, I developed my conceptual framework of medical providers’ treatment habitus (i.e., medical providers’ dispositions towards caring for PLWH-SU) and estimated a typology of treatment habitus using survey data from 258 medical providers in Miami, Florida, Atlanta, Georgia, and the District of Columbia. My analyses show that among this sample of medical providers, there are four types of treatment habitus towards caring for PLWH-SU, and treatment habitus is associated with multi-level factors (e.g., providers’ race, study site, receipt of substance use disorder training). To further explore how medical providers came to develop and how they understand their own treatment habitus, I conducted conversational interviews with 36 medical providers who had completed the abovementioned survey. These interviews revealed medical providers exhibit a spectrum of treatment habitus that is distinguishable by their intentions (person-centered vs. provider-centered) and their methods (informative vs. directive). The interviews also revealed that there are discrepancies in how medical providers spoke about PLWH-SU and how they described their practices towards caring for PLWH-SU. Specifically, although most providers used negative terms to refer to PLWH-SU, the stigmatizing language was almost never accompanied by recollections of stigmatizing behaviors during clinical interactions with PLWH-SU. Taken together, this dissertation expanded on current knowledge about not only how medical providers act when caring for PLWH-SU, but also why they act the ways they do. Findings from this study contribute to an understudied area of HIV and substance use research and provide insights for the development of novel provider-based interventions that can improve the health of this vulnerable and marginalized population.

This dissertation seeks to incrementally explain the impact of individual, interpersonal, and environmental levels of risk upon HIV/STI incidents among heterosexual African American serodiscordant couples residing in four metropolitan cities. Using archival data from a cluster-RCT (Project EBAN) and governmental surveillance reports, analytic methods that can model heterogeneous pathways within and across each level of risk were used. Findings from this dissertation revealed unique patterns and pathways via which African American females in serodiscordant relationships contracted HIV/STI.