AIDS: structure, replication and spread of the AIDS virus

AIDS (Acquired Immune Deficiency Syndrome)was first, described as a new disease of humans in 1981. The origins of the disease are controversial. AIDS is caused by a retrovirus, a type of virus which rarely attacks human cells. The first virus of this type recorded in humans is reponsible for a type of leukaemia and was identified in 1978. AIDS is thus the third type of human retrovirus to be discovered and hence, is referred to as T-lymphotrophic virus III (HTLV-III). For viruses to replicate, they have to invade a host cell which in this case is a T4-lymphocyte, a type of white blood cell that regulates the immune system. The problems of the disease result directly from the death of these cells. As a consequence, the immune system is compromised leading to a number of opportunistic secondary infections and other disorders.

The production, harvest and adsorptive recovery of an infectious herpes simplex virus vaccine

At present there is not a reliable vaccine against herpes virus. Viral protein vaccines as yet have proved unsuccessful to meet the challenge of raising an appropriate immune response. Cantab Pharmaceuticals has produced a virus vaccine that can undergo one round of replication in the recipient in order to produce a more specific immune reaction. This virus is called Disabled Infectious Single Cycle Herpes Simplex Virus (DISC HSV) which has been derived by deleting the essential gH gene from a type 2 herpes virus. This vaccine has been proven to be effective in animal studies. Existing methods for the purification of viruses rely on laboratory techniques and for vaccine production would be on a far too small a scale. There is therefore a need for new virus purification methods to be developed in order to meet these large scale needs. An integrated process for the manufacture of a purified recombinant DISC HSV is described. The process involves culture of complementing Vero (CR2) cells, virus infection and manufacture, virus harvesting and subsequent downstream processing. The identification of suitable growth parameters for the complementing cell line and optimal limes for both infection and harvest are addressed. Various traditional harvest methods were investigated and found not to be suitable for a scaled up process. A method of harvesting, that exploits the elution of cell associated viruses by the competitive binding of exogenous heparin to virus envelope gC proteins, is described and is shown to yield significantly less contaminated process streams than sonication or osmotic approaches that involve cell rupture (with> 10-fold less complementing cell protein). High concentrations of salt (>0.8M NaCl) exhibit the same effect, although the high osmotic strength ruptures cells and increase the contamination of the process stream. This same heparin-gC protein affinity interaction is also shown to provide an efficient adsorptive purification procedure for herpes viruses which avoids the need to pre-treat the harvest material, apart from clarification, prior to chromatography. Subsequent column eluates provide product fractions with a 100-fold increase in virus titre and low levels of complementing cell protein and DNA (0.05 pg protein/pfu and 1.2 x 104 pg DNA/pfu respectively).

Interaction of the parasitic angiosperm {\it Cassytha filiformis\/} L. (Lauraceae) with the exotic {\it Schinus terebinthifolius\/} Raddi (Anacardiaceae) in southern Florida

Brazilian pepper (Schinus terebinthifolius) is an exotic shrub or small tree that has become well established as an invasive and highly competitive species through much of southern Florida. Love vine (Cassytha filiformis), a native parasitic plant, was noted parasitizing Brazilian pepper, apparently affecting its health. The objective of this study was to investigate the nature of this parasitic interaction in southern Florida. Brazilian pepper populations were studied to determine whether parasitism by love vine may affect growth and reproduction. Anatomical studies of love vine parasitizing Brazilian pepper determined physical aspects of the parasitic interaction at the cell and tissue level. Physiological aspects of this interaction were investigated to help describe love vine resource acquisition as a parasite on host Brazilian pepper plants, and as an autotrophic plant. An investigation of ecological aspects of this parasitic interaction was done to determine whether physical or biological aspects of habitats may contribute to love vine parasitism on Brazilian pepper. These studies indicated that: (1) parasitism by love vine significantly decreased growth and reproduction of Brazilian pepper plants; (2) anatomical and physiological investigations indicated that love vine was primarily a xylem parasite on Brazilian pepper, but that some assimilated carbon nutrients may also be acquired from the host; (3) love vine is autotrophic (i.e., hemiparasitic), but is totally dependent on its host for necessary resources; (4) the occurrence of love vine parasitism on Brazilian pepper is mediated by physical characters of the biological community. ^

Interaction of the parasitic angiosperm cassytha filiformis L. (Lauraceae) with the exotic schinus terebinthifolius RADDI (Anacardiaceae) in southern Florida

Brazilian pepper (Schinus terebinthifolius) is an exotic shrub or small tree that has become well established as an invasive and highly competitive species through much of southern Florida. Love vine (Cassytha filiformis), a native parasitic plant, was noted parasitizing Brazilian pepper, apparently affecting its health. The objective of this study was to investigate the nature of this parasitic interaction in southern Florida. Brazilian pepper populations were studied to determine whether parasitism by love vine may affect growth and reproduction. Anatomical studies of love vine parasitizing Brazilian pepper determined physical aspects of the parasitic interaction at the cell and tissue level. Physiological aspects of this interaction were investigated to help describe love vine resource acquisition as a parasite on host Brazilian pepper plants, and as an autotrophic plant. An investigation of ecological aspects of this parasitic interaction was done to determine whether physical or biological aspects of habitats may contribute to love vine parasitism on Brazilian pepper. These studies indicated that: 1) parasitism by love vine significantly decreased growth and reproduction of Brazilian pepper plants; 2) anatomical and physiological investigations indicated that love vine was primarily a xylem parasite on Brazilian pepper, but that some assimilated carbon nutrients may also be acquired from the host; 3) love vine is autotrophic (i. e., hemiparasitic), but is totally dependent on its host for necessary resources; 4) the occurrence of love vine parasitism on Brazilian pepper is mediated by physical characters of the biological community.

Rôle de la protéine virale Vpu dans le cycle de multiplication du virus de l'immunodéficience humaine de type 1 (VIH-1)

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The Irish paradigm on the natural progression of hepatitis C virus infection: an investigation in a homogeneous patient population infected with HCV 1b (Review)

The aetiological agent of chronic hepatitis C is the hepatitis C virus. The hepatitis C virus is spread by parenteral transmission of body fluids, primarily blood or blood products. In 1989, after more than a decade of research, HCV was isolated and characterised. The hepatitis C viral genome is a positive-sense, single-stranded RNA molecule approximately 9.4 kb in length, which encodes a polyprotein of about 3100 amino acids. There are 6 main genotypes of HCV, each further stratified by subtype. In 1994, a cohort of women was identified in Ireland as having been iatrogenically exposed to the hepatitis C virus. The women were all young and exposed as a consequence of the receipt of HCV 1b contaminated anti-D immunoglobulin. The source of the infection was identified as an acutely infected female. As part of a voluntary serological screening programme involving 62,667 people, 704 individuals were identified as seropositive for exposure to the hepatitis C virus; 55.4% were found to be positive for the viral genome 17 years after exposure. Of these women 98% had evidence of inflammation, but suprisingly, a remarkable 49% showed no evidence of fibrosis. Clinicopathology and virological analysis has identified associations between viral load and the histological activity index for inflammation, and, between inflammation and levels of the liver enzyme alanine aminotransferase. Infection at a younger age appears to protect individuals from progression to advanced liver disease. Molecular analyses of host immunogenetic elements shows that particular class II human leukocyte associated antigen alleles are associated with clearance of the hepatitis C virus. Additional class II alleles have been identified that are associated with stable viraemia over an extended period of patient follow-up. Although, investigation of large untreated homogeneous cohorts is likely to become more difficult, as the efficacy of anti-viral therapy improves, further investigation of host and viral factors that influence disease progression will help provide an evidence based approach were realistic expectations regarding patient prognosis can be ascertained.

RNA Virus Evolution: a Cross-scale, Disease Dynamic Perspective

RNA viruses are an important cause of global morbidity and mortality. The rapid evolutionary rates of RNA virus pathogens, caused by high replication rates and error-prone polymerases, can make the pathogens difficult to control. RNA viruses can undergo immune escape within their hosts and develop resistance to the treatment and vaccines we design to fight them. Understanding the spread and evolution of RNA pathogens is essential for reducing human suffering. In this dissertation, I make use of the rapid evolutionary rate of viral pathogens to answer several questions about how RNA viruses spread and evolve. To address each of the questions, I link mathematical techniques for modeling viral population dynamics with phylogenetic and coalescent techniques for analyzing and modeling viral genetic sequences and evolution. The first project uses multi-scale mechanistic modeling to show that decreases in viral substitution rates over the course of an acute infection, combined with the timing of infectious hosts transmitting new infections to susceptible individuals, can account for discrepancies in viral substitution rates in different host populations. The second project combines coalescent models with within-host mathematical models to identify driving evolutionary forces in chronic hepatitis C virus infection. The third project compares the effects of intrinsic and extrinsic viral transmission rate variation on viral phylogenies.

Hepatitis C virus modulation of lipid and autophagy pathways

Hepatitis C virus [HCV] infects 170 million people worldwide. We investigated interactions between HCV proteins and cellular proteins involved in autophagy and lipid metabolism. We sought to develop an infection model using patient derived human serum containing HCV and human hepatocytes, Huh7 cells. Using the model, we have shown intracellular expression of incoming HCV RNA (5′ UTR region and region spanning the E1/E2 glycoproteins), expression of the HCV proteins, core and NS5B, and a cellular response to HCV infection. These data suggests this model can be used to analyse the early stage of HCV infection. HCV utilises the autophagy pathway to both establish infection and to complete its life cycle. We investigated HCV interaction with the early stage autophagy protein ATG5. We found that although ATG5 mRNA is unchanged in HCV infected cells, protein expression of ATG5 is significantly upregulated. These data indicated HCV controls the post-transcriptional regulation of ATG5. We used the upstream open reading frame (uORF) and the 5′ UTR region of ATG5 to examine the post-transcriptional regulation. Our data suggest HCV RNA replication either directly or indirectly causes post-transcriptional regulation of the early autophagy protein, ATG5 in a 5′ UTR and uORF independent manner. HCV infection leads to an increase in SREBP controlled genes e.g. HMG-CoA Reductase, cholesterol, LDL and fatty acid synthesis. We hypothesised that HCV infection causes the activation of SREBP pathway by interacting directly or indirectly with proteins involved in the initiation of the pathway. We sought to determine if HCV interacts with SCAP or INSIG. We confirmed a change in LD distribution and HMG-CoA reductase activity as a result of HCV RNA replication. Significantly, we show SCAP protein expression was also altered during HCV RNA replication and HCV core protein possibly interacts with SCAP.

Rôle de la protéine virale Vpu dans le cycle de multiplication du virus de l'immunodéficience humaine de type 1 (VIH-1)

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Superinfection exclusion and the long-term survival of honey bees in Varroa-infested colonies

Over the past 50 years, many millions of European honey bee (Apis mellifera) colonies have died as the ectoparasitic mite, Varroa destructor, has spread around the world. Subsequent studies have indicated that the mite’s association with a group of RNA viral pathogens (Deformed Wing Virus, DWV) correlates with colony death. Here, we propose a phenomenon known as superinfection exclusion that provides an explanation of how certain A. mellifera populations have survived, despite Varroa infestation and high DWV loads. Next-generation sequencing has shown that a non-lethal DWV variant ‘type B’ has become established in these colonies and that the lethal ‘type A’ DWV variant fails to persist in the bee population. We propose that this novel stable host-pathogen relationship prevents the accumulation of lethal variants, suggesting that this interaction could be exploited for the development of an effective treatment that minimises colony losses in the future.

Superinfection exclusion and the long-term survival of honey bees in Varroa-infested colonies

Over the past 50 years, many millions of European honey bee (Apis mellifera) colonies have died as the ectoparasitic mite, Varroa destructor, has spread around the world. Subsequent studies have indicated that the mite’s association with a group of RNA viral pathogens (Deformed Wing Virus, DWV) correlates with colony death. Here, we propose a phenomenon known as superinfection exclusion that provides an explanation of how certain A. mellifera populations have survived, despite Varroa infestation and high DWV loads. Next-generation sequencing has shown that a non-lethal DWV variant ‘type B’ has become established in these colonies and that the lethal ‘type A’ DWV variant fails to persist in the bee population. We propose that this novel stable host-pathogen relationship prevents the accumulation of lethal variants, suggesting that this interaction could be exploited for the development of an effective treatment that minimises colony losses in the future.

von Hippel Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin-proteasome pathway. Here, we identify von Hippel-Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel-Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin-VHL-proteasome pathway in the integration-transcription transition of the viral replication cycle.

Nuclear import of HIV-1 integrase is inhibited in vitro by styrylquinoline derivatives

Nuclear import of HIV-1 preintegration complexes (PICs) allows the virus to infect nondividing cells. Integrase (IN), the PIC-associated viral enzyme responsible for the integration of the viral genome into the host cell DNA, displays karyophilic properties and has been proposed to participate to the nuclear import of the PIC. Styrylquinolines (SQs) have been shown to block viral replication at nontoxic concentrations and to inhibit IN 3'-processing activity in vitro by competing with the DNA substrate binding. However, several lines of evidence suggested that SQs could have a postentry, preintegrative antiviral effect in infected cells. To gain new insights on the mechanism of their antiviral activity, SQs were assayed for their ability to affect nuclear import of HIV-1 IN and compared with the effect of a specific strand transfer inhibitor. Using an in vitro transport assay, we have previously shown that IN import is a saturable mechanism, thus showing that a limiting cellular factor is involved in this process. We now demonstrate that SQs specifically and efficiently inhibit in vitro nuclear import of IN without affecting other import pathways, whereas a specific strand transfer inhibitor does not affect IN import. These data suggest that SQs not only inhibit IN-DNA interaction but would also inhibit the interaction between IN and the cellular factor required for its nuclear import.

Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Drosophila Adaptation to Viral Infection through Defensive Symbiont Evolution

Microbial symbionts can modulate host interactions with biotic and abiotic factors. Such interactions may affect the evolutionary trajectories of both host and symbiont. Wolbachia protects Drosophila melanogaster against several viral infections and the strength of the protection varies between variants of this endosymbiont. Since Wolbachia is maternally transmitted, its fitness depends on the fitness of its host. Therefore, Wolbachia populations may be under selection when Drosophila is subjected to viral infection. Here we show that in D. melanogaster populations selected for increased survival upon infection with Drosophila C virus there is a strong selection coefficient for specific Wolbachia variants, leading to their fixation. Flies carrying these selected Wolbachia variants have higher survival and fertility upon viral infection when compared to flies with the other variants. These findings demonstrate how the interaction of a host with pathogens shapes the genetic composition of symbiont populations. Furthermore, host adaptation can result from the evolution of its symbionts, with host and symbiont functioning as a single evolutionary unit.