1. The document discusses research into visualizing the interaction between the HIV viral envelope and capsid protein using freeze-fracture electron microscopy.
2. The goal is to better understand how the viral envelope attaches to the capsid, which could lead to methods to disrupt this process and inhibit HIV infection.
3. Introducing a premature stop codon in the Gag protein will arrest viral bud release, allowing visualization of the arrested viral buds and interaction between the envelope and capsid.
Adenoviral vectors are modified adenoviruses that can deliver genetic material into host cells. Adenoviruses are medium-sized, non-enveloped viruses containing double-stranded DNA. They can efficiently transfer DNA/RNA into cells and have been used to construct viral vectors. Wild type adenoviruses are modified by deleting non-essential genes and adding exogenous genetic material to create viral vectors. Three generations of adenoviral vectors have been developed for gene therapy, with later generations having improved safety profiles and ability to carry larger DNA payloads.
This document discusses adenoviral cloning vectors. It begins by defining a cloning vector as a small piece of DNA that can be stably maintained in an organism and have foreign DNA inserted into it for cloning purposes. It then discusses viral vectors, noting that they are commonly used to deliver genetic material into cells through transduction. The document focuses on properties of viral vectors, specifically safety features and targeting abilities. It provides details on adenoviruses, noting they can efficiently transfer genes, their structure, applications in gene therapy and vaccination, and their DNA genome capacity. Adeno-associated viruses are also mentioned as attractive for gene therapy due to mild immune response.
1) There are various COVID-19 vaccine candidates that target different parts of the SARS-CoV-2 spike protein, including the full-length spike protein and the receptor-binding domain (RBD).
2) Some research suggests the RBD may be a better target than the full-length spike protein due to concerns that the full protein could potentially cause immune pathology.
3) However, vaccines using the full-length spike protein aim to better mimic the natural infection and induce antibodies and T-cell responses against multiple epitopes on the spike protein.
The document discusses coronaviruses and their variants. It begins with a brief history of coronaviruses, noting their discovery in the 1930s and their naming due to their appearance under electron microscopy. It then discusses taxonomy and the MRCA of coronaviruses dating back to 8000 BCE. It provides details on SARS-CoV-1 and SARS-CoV-2, including their structures, receptors, and differences in infectiousness. It then summarizes transmission patterns and clinical symptoms of COVID-19 before discussing immune evasion strategies of coronaviruses and challenges to vaccine development. It concludes with an overview of coronavirus variants and classification systems.
This document provides information on the phenotype and genotype of COVID-19. It discusses that coronaviruses are RNA viruses that cause respiratory infections in humans. COVID-19 is caused by a novel betacoronavirus. The virus particle is spherical with spikes and enters cells by binding to the ACE2 receptor. It contains a positive-sense RNA genome that is translated and replicated via a replicase-transcriptase complex to make structural proteins and more virus particles.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Methodology for development of defined deletion mutant vaccine for salmonellosisBhoj Raj Singh
1. The document discusses methodology for developing defined deletion mutant vaccines for salmonellosis. Common gene targets for deletion mutagenesis in Salmonella include galE, aroA, cya, crp, PhoP, PhoQ and others.
2. Methods for producing defined deletion mutants include using suicide plasmids for homologous recombination to replace target genes with truncated versions lacking internal sequences. Several techniques for making precise deletions are described.
3. Safety and efficacy testing in animal models is required before field trials, including testing for attenuation, immunogenicity, stability of mutations, safety, and ability to prevent disease caused by wild-type strains.
The document discusses various viral delivery systems for gene therapy. It notes four major problems with current gene therapy: short-lived expression, immune response, issues with viral vectors, and difficulties treating multigene disorders. Common viral vectors discussed include retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. Each vector type has advantages and limitations for gene delivery applications. Retroviruses can only target dividing cells, while lentiviruses can target both dividing and non-dividing cells. Adenoviral vectors have high transduction efficiency but also elicit strong immune responses. AAV vectors allow long-term expression but have a small cargo capacity.
Adenoviral vectors are modified adenoviruses that can deliver genetic material into host cells. Adenoviruses are medium-sized, non-enveloped viruses containing double-stranded DNA. They can efficiently transfer DNA/RNA into cells and have been used to construct viral vectors. Wild type adenoviruses are modified by deleting non-essential genes and adding exogenous genetic material to create viral vectors. Three generations of adenoviral vectors have been developed for gene therapy, with later generations having improved safety profiles and ability to carry larger DNA payloads.
This document discusses adenoviral cloning vectors. It begins by defining a cloning vector as a small piece of DNA that can be stably maintained in an organism and have foreign DNA inserted into it for cloning purposes. It then discusses viral vectors, noting that they are commonly used to deliver genetic material into cells through transduction. The document focuses on properties of viral vectors, specifically safety features and targeting abilities. It provides details on adenoviruses, noting they can efficiently transfer genes, their structure, applications in gene therapy and vaccination, and their DNA genome capacity. Adeno-associated viruses are also mentioned as attractive for gene therapy due to mild immune response.
1) There are various COVID-19 vaccine candidates that target different parts of the SARS-CoV-2 spike protein, including the full-length spike protein and the receptor-binding domain (RBD).
2) Some research suggests the RBD may be a better target than the full-length spike protein due to concerns that the full protein could potentially cause immune pathology.
3) However, vaccines using the full-length spike protein aim to better mimic the natural infection and induce antibodies and T-cell responses against multiple epitopes on the spike protein.
The document discusses coronaviruses and their variants. It begins with a brief history of coronaviruses, noting their discovery in the 1930s and their naming due to their appearance under electron microscopy. It then discusses taxonomy and the MRCA of coronaviruses dating back to 8000 BCE. It provides details on SARS-CoV-1 and SARS-CoV-2, including their structures, receptors, and differences in infectiousness. It then summarizes transmission patterns and clinical symptoms of COVID-19 before discussing immune evasion strategies of coronaviruses and challenges to vaccine development. It concludes with an overview of coronavirus variants and classification systems.
This document provides information on the phenotype and genotype of COVID-19. It discusses that coronaviruses are RNA viruses that cause respiratory infections in humans. COVID-19 is caused by a novel betacoronavirus. The virus particle is spherical with spikes and enters cells by binding to the ACE2 receptor. It contains a positive-sense RNA genome that is translated and replicated via a replicase-transcriptase complex to make structural proteins and more virus particles.
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Methodology for development of defined deletion mutant vaccine for salmonellosisBhoj Raj Singh
1. The document discusses methodology for developing defined deletion mutant vaccines for salmonellosis. Common gene targets for deletion mutagenesis in Salmonella include galE, aroA, cya, crp, PhoP, PhoQ and others.
2. Methods for producing defined deletion mutants include using suicide plasmids for homologous recombination to replace target genes with truncated versions lacking internal sequences. Several techniques for making precise deletions are described.
3. Safety and efficacy testing in animal models is required before field trials, including testing for attenuation, immunogenicity, stability of mutations, safety, and ability to prevent disease caused by wild-type strains.
The document discusses various viral delivery systems for gene therapy. It notes four major problems with current gene therapy: short-lived expression, immune response, issues with viral vectors, and difficulties treating multigene disorders. Common viral vectors discussed include retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. Each vector type has advantages and limitations for gene delivery applications. Retroviruses can only target dividing cells, while lentiviruses can target both dividing and non-dividing cells. Adenoviral vectors have high transduction efficiency but also elicit strong immune responses. AAV vectors allow long-term expression but have a small cargo capacity.
The document summarizes recent research on SARS Coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19. It describes the virus's life cycle, including its structure, genome, and replication process. Advances in molecular biology and reverse genetics have increased understanding of coronavirus genetics, adaptation, and pathogenesis. While previous coronaviruses like SARS and MERS jumped from animals to humans, the COVID-19 pandemic demonstrates SARS-CoV-2's ability to transmit efficiently between humans. Further research aims to define the viral and host factors driving transmission and disease.
- The mRNA-1273 vaccine induced robust antibody and neutralizing responses against SARS-CoV-2 in all participants after the first dose. A second dose led to even stronger responses, especially in the 100 μg and 250 μg groups. Systemic side effects were mostly mild or moderate. This interim report provides support for mRNA-1273 as a promising COVID-19 vaccine candidate warranting further development and evaluation.
This document summarizes safety considerations for DNA vaccines. It discusses safety issues related to the genetic elements of DNA vaccines, including the potential for antibiotic resistance gene transfer and germline integration. It also discusses safety concerns regarding the microbial production host, such as endotoxin production and genetic instability. The document proposes strategies to improve safety, including using replication origins with narrow host range, non-antibiotic selection markers, artificial promoter and signal sequences with low human homology, and gram-positive microbial hosts like Lactococcus lactis that do not produce endotoxins.
Using SARS-CoV-2 to Teach Physiology and ScienceInsideScientific
Join Dr. Dee Silverthorn for a discussion on how the sudden appearance of the global pandemic of COVID-19 provides a unique opportunity to show students science in action as researchers and healthcare professionals around the world scramble to understand the virus and its effects on the human body. This is the third webinar in this 4-part series on how science education has evolved in the face of new challenges.
In this presentation we will explore some of the ways that we can incorporate today’s headlines into the curriculum by discussing the pathophysiology and pathology of SARS-CoV-2 infection and how it demonstrates the integration of body function across multiple organ systems. Teaching about the coronavirus pandemic also creates opportunities to have students critically analyze research studies and news reports, and to discuss ethical dilemmas such as the distribution of limited amounts of vaccine or the triage of critically ill patients when lifesaving equipment is limited. One important goal of teaching about the coronavirus pandemic is to have students learn to tolerate ambiguity, and to understand that today’s “facts” are simply our best models of what we know.
The document discusses the structure, replication, pathogenesis, and treatment of three viruses:
1) Adenovirus has a medium sized double stranded DNA genome, naked capsid, and causes respiratory or gastrointestinal infections through direct cell damage. It is treated with live military vaccines.
2) Parvovirus has a small single stranded DNA genome, replicates in host cell nuclei and depends on host cell cycle, and causes fifth disease and aplastic crisis in children. No specific treatment exists.
3) Polyomavirus has a small circular double stranded DNA genome, replicates in host cell nuclei and depends on the host, can cause PML with immune compromise, and may be treated with cidofovir.
Researchers have discovered a novel maturation mechanism employed by the hepatitis B virus (HBV) that improves its ability to infect liver cells. HBV particles are released from infected cells in an "immature" form that cannot bind to liver cells. Over time, the particles undergo a slow maturation process where they switch to a "mature" form able to bind to liver cells. This allows the particles to circulate in the bloodstream until reaching the liver, where they can then infect cells. This explains how HBV can specifically target liver cells for infection even when few virus particles enter the body. The finding could help develop new drugs that inhibit the maturation process and prevent HBV from becoming infectious.
This document summarizes research into developing an anti-HIV vaccine using nucleoside-modified mRNA encoding envelope proteins. Key points:
- Researchers developed mRNA vaccines optimized for protein expression by incorporating modified nucleosides, UTRs, caps, and tails to generate HIV envelope proteins.
- Mice were primed with intradermal mRNA followed by an intramuscular protein boost to achieve strong T cell and B cell responses.
- The mRNA vaccine induced high levels of IFN-γ, TNF-α, IL-2 in antigen-specific T cells and antibody titers, demonstrating the potential of nucleoside-modified mRNA vaccines for infectious diseases like HIV.
The concept of transferring genes to tissues for clinical applications has been discussed for nearly half a century, but the ability to manipulate genetic material via recombinant DNA technology has brought this goal to reality. ‘Gene Therapy’ covers both the research and clinical applications of the new genetic therapy techniques currently being developed. The application of molecular biology has revolutionized researchers understanding of many diseases and has been readily applied for diagnostic purposes. Now-a-day this is originally conceived as a way to treat life-threatening disorders (inborn errors, cancers) refractory to conventional treatment, gene therapy now is considered for many non–life-threatening conditions, including those adversely affecting a patient’s quality of life. The lack of suitable treatment has become a rational basis for extending the scope of gene therapy. It is not very far, the justifiable optimism that with increased biotechnological improvement, gene therapy will become a standard part of clinical practice.
Viruses and Cancer: Introduction to OncovirusesAparna Dubey
The presentation describes about Oncoviruses or the cancer causing viruses with emphasis on HPV ( Human papillomavirus ) responsible for Cervical Cancer.
In this presentation, I talked about the new mRNA vaccine that is authorized for the prevention of coronavirus infection.
mRNA 1273 is developed by Moderna in the US and has shown almost 94% effectiveness
Watch the slideshow for a better understanding: https://youtu.be/frmGwCEtDnM
1. Learn how mRNA vaccines work.
2. Learn challenges in making HIV vaccines.
3. Learn about the advantage of mRNA vaccines in HIV
4. Learn about trials.
CD Genomics provides viral genome sequencing services using Illumina and PacBio platforms to generate high-quality de novo assemblies of large viral genomes at low cost. Viruses are the most abundant biological entities on Earth and significantly impact living organisms, but less than 0.01% have been sequenced. Viral genome sequencing holds potential for understanding virus diversity, ecology, adaptation, and evolution and enabling prediction of emerging infectious diseases. CD Genomics' sequencing services include no reference sequence requirement, complete characterization of viral genomes, identification of minor variants, and cost-effective high-throughput sequencing with fast turnaround of high quality data.
This document discusses DNA viruses and viral vectors. It begins by introducing viruses and their classification based on host, morphology, genome, and structures. It then describes the Baltimore classification system for viruses. Viral vectors are discussed as effective means of gene transfer that can be manipulated to express therapeutic genes. Several virus types being investigated for gene delivery are described, including retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. Key properties of viral vectors like safety, toxicity, stability, and cell specificity are outlined. Specific types of viral vectors - retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors - are then characterized.
This patent document describes the isolation and characterization of a novel human coronavirus (SARS-CoV) that is the causative agent of severe acute respiratory syndrome (SARS). It provides the nucleic acid sequence of the SARS-CoV genome and the amino acid sequences of its open reading frames. Methods are described for using these molecules to detect SARS-CoV and detect infections. Immune stimulatory compositions are also provided, along with methods for their use.
presentation on the molecular biology of Coronaviruses which include taxonomy, history of the viruses, various proteins present in virus, their structure, importance, roles, the life cycle of virus, infection process, the process of disease development, the pathogenicity of virus, replication and translation process in coronaviruses, possible sites for vaccine development, available treatments, cures and drugs, and various studies regarding coronaviruses infection and cures.
Creative Biolabs has extensive experience in coronavirus research. Provide comprehensive high-quality coronavirus (SARS-CoV-2, SARS-CoV, MERS-CoV, etc.) related services and products.
This slide provides a brief introduction to SARS-CoV-2. If you need more knowledge, products and services related to SARS-CoV-2, please follow us.
The document is a United States patent describing methods for producing recombinant coronavirus particles. Specifically, it describes developing a helper cell that contains: 1) a coronavirus permissive cell; 2) a coronavirus replicon RNA containing a heterologous sequence and packaging signal but lacking a structural protein gene; and 3) a separate helper RNA encoding the missing structural protein. When these components are expressed in the cell, coronavirus particles are assembled that contain the heterologous RNA sequence but cannot replicate without the helper RNA. The patent claims compositions and methods for making and using these viral particles to deliver heterologous genes.
This document summarizes research on plant viral suppressors of RNA silencing. It begins by providing background on how RNA silencing was discovered as an antiviral defense mechanism in plants. Most plant viruses encode suppressor proteins that interfere with this silencing pathway. The review then describes the major functional assays that are used to identify these viral suppressor proteins, including transient expression assays using Agrobacterium infiltration and assays to test whether viral infection or expression of a candidate protein can reverse an established silenced phenotype. It focuses on the currently known viral suppressors from different plant virus families and the steps in the RNA silencing pathway that they target.
SARS-CoV-2 was first identified in Wuhan, China in late 2019 and has since spread to over 200 countries. It belongs to the coronavirus family and causes the disease COVID-19. The virus enters cells through the ACE2 receptor and replicates its RNA and proteins before being released to infect other cells. Potential treatment strategies include using existing antiviral drugs, developing specific drugs that target the virus genome or proteins, and enhancing immune responses. Ongoing research focuses on antibody development, diagnostic assays, vaccines, and other medical countermeasures to treat and prevent the spread of SARS-CoV-2.
This study aimed to optimize norovirus GI genotyping primers and apply them to characterize norovirus GI diversity in clinical and environmental samples from South Africa. Five norovirus GI genotypes were found circulating between 2015-2016, with GI.4 being the most prevalent in 63.2% of samples. The primers were optimized to improve genotyping of viruses from sewage samples. National and regional strain clusters were identified, adding to understanding of norovirus genetics and transmission globally.
This document provides information on performance evaluation methods for forensic specialists. It discusses 12 common methods: management by objectives, critical incident method, behaviorally anchored rating scales, behavioral observation scales, 360 degree appraisal, and checklist and weighted checklist method. For each method, it provides a definition and overview, as well as advantages and disadvantages. The goal is to help choose effective approaches for evaluating a forensic specialist's job performance.
The document summarizes recent research on SARS Coronavirus-2 (SARS-CoV-2), the virus that causes COVID-19. It describes the virus's life cycle, including its structure, genome, and replication process. Advances in molecular biology and reverse genetics have increased understanding of coronavirus genetics, adaptation, and pathogenesis. While previous coronaviruses like SARS and MERS jumped from animals to humans, the COVID-19 pandemic demonstrates SARS-CoV-2's ability to transmit efficiently between humans. Further research aims to define the viral and host factors driving transmission and disease.
- The mRNA-1273 vaccine induced robust antibody and neutralizing responses against SARS-CoV-2 in all participants after the first dose. A second dose led to even stronger responses, especially in the 100 μg and 250 μg groups. Systemic side effects were mostly mild or moderate. This interim report provides support for mRNA-1273 as a promising COVID-19 vaccine candidate warranting further development and evaluation.
This document summarizes safety considerations for DNA vaccines. It discusses safety issues related to the genetic elements of DNA vaccines, including the potential for antibiotic resistance gene transfer and germline integration. It also discusses safety concerns regarding the microbial production host, such as endotoxin production and genetic instability. The document proposes strategies to improve safety, including using replication origins with narrow host range, non-antibiotic selection markers, artificial promoter and signal sequences with low human homology, and gram-positive microbial hosts like Lactococcus lactis that do not produce endotoxins.
Using SARS-CoV-2 to Teach Physiology and ScienceInsideScientific
Join Dr. Dee Silverthorn for a discussion on how the sudden appearance of the global pandemic of COVID-19 provides a unique opportunity to show students science in action as researchers and healthcare professionals around the world scramble to understand the virus and its effects on the human body. This is the third webinar in this 4-part series on how science education has evolved in the face of new challenges.
In this presentation we will explore some of the ways that we can incorporate today’s headlines into the curriculum by discussing the pathophysiology and pathology of SARS-CoV-2 infection and how it demonstrates the integration of body function across multiple organ systems. Teaching about the coronavirus pandemic also creates opportunities to have students critically analyze research studies and news reports, and to discuss ethical dilemmas such as the distribution of limited amounts of vaccine or the triage of critically ill patients when lifesaving equipment is limited. One important goal of teaching about the coronavirus pandemic is to have students learn to tolerate ambiguity, and to understand that today’s “facts” are simply our best models of what we know.
The document discusses the structure, replication, pathogenesis, and treatment of three viruses:
1) Adenovirus has a medium sized double stranded DNA genome, naked capsid, and causes respiratory or gastrointestinal infections through direct cell damage. It is treated with live military vaccines.
2) Parvovirus has a small single stranded DNA genome, replicates in host cell nuclei and depends on host cell cycle, and causes fifth disease and aplastic crisis in children. No specific treatment exists.
3) Polyomavirus has a small circular double stranded DNA genome, replicates in host cell nuclei and depends on the host, can cause PML with immune compromise, and may be treated with cidofovir.
Researchers have discovered a novel maturation mechanism employed by the hepatitis B virus (HBV) that improves its ability to infect liver cells. HBV particles are released from infected cells in an "immature" form that cannot bind to liver cells. Over time, the particles undergo a slow maturation process where they switch to a "mature" form able to bind to liver cells. This allows the particles to circulate in the bloodstream until reaching the liver, where they can then infect cells. This explains how HBV can specifically target liver cells for infection even when few virus particles enter the body. The finding could help develop new drugs that inhibit the maturation process and prevent HBV from becoming infectious.
This document summarizes research into developing an anti-HIV vaccine using nucleoside-modified mRNA encoding envelope proteins. Key points:
- Researchers developed mRNA vaccines optimized for protein expression by incorporating modified nucleosides, UTRs, caps, and tails to generate HIV envelope proteins.
- Mice were primed with intradermal mRNA followed by an intramuscular protein boost to achieve strong T cell and B cell responses.
- The mRNA vaccine induced high levels of IFN-γ, TNF-α, IL-2 in antigen-specific T cells and antibody titers, demonstrating the potential of nucleoside-modified mRNA vaccines for infectious diseases like HIV.
The concept of transferring genes to tissues for clinical applications has been discussed for nearly half a century, but the ability to manipulate genetic material via recombinant DNA technology has brought this goal to reality. ‘Gene Therapy’ covers both the research and clinical applications of the new genetic therapy techniques currently being developed. The application of molecular biology has revolutionized researchers understanding of many diseases and has been readily applied for diagnostic purposes. Now-a-day this is originally conceived as a way to treat life-threatening disorders (inborn errors, cancers) refractory to conventional treatment, gene therapy now is considered for many non–life-threatening conditions, including those adversely affecting a patient’s quality of life. The lack of suitable treatment has become a rational basis for extending the scope of gene therapy. It is not very far, the justifiable optimism that with increased biotechnological improvement, gene therapy will become a standard part of clinical practice.
Viruses and Cancer: Introduction to OncovirusesAparna Dubey
The presentation describes about Oncoviruses or the cancer causing viruses with emphasis on HPV ( Human papillomavirus ) responsible for Cervical Cancer.
In this presentation, I talked about the new mRNA vaccine that is authorized for the prevention of coronavirus infection.
mRNA 1273 is developed by Moderna in the US and has shown almost 94% effectiveness
Watch the slideshow for a better understanding: https://youtu.be/frmGwCEtDnM
1. Learn how mRNA vaccines work.
2. Learn challenges in making HIV vaccines.
3. Learn about the advantage of mRNA vaccines in HIV
4. Learn about trials.
CD Genomics provides viral genome sequencing services using Illumina and PacBio platforms to generate high-quality de novo assemblies of large viral genomes at low cost. Viruses are the most abundant biological entities on Earth and significantly impact living organisms, but less than 0.01% have been sequenced. Viral genome sequencing holds potential for understanding virus diversity, ecology, adaptation, and evolution and enabling prediction of emerging infectious diseases. CD Genomics' sequencing services include no reference sequence requirement, complete characterization of viral genomes, identification of minor variants, and cost-effective high-throughput sequencing with fast turnaround of high quality data.
This document discusses DNA viruses and viral vectors. It begins by introducing viruses and their classification based on host, morphology, genome, and structures. It then describes the Baltimore classification system for viruses. Viral vectors are discussed as effective means of gene transfer that can be manipulated to express therapeutic genes. Several virus types being investigated for gene delivery are described, including retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. Key properties of viral vectors like safety, toxicity, stability, and cell specificity are outlined. Specific types of viral vectors - retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors - are then characterized.
This patent document describes the isolation and characterization of a novel human coronavirus (SARS-CoV) that is the causative agent of severe acute respiratory syndrome (SARS). It provides the nucleic acid sequence of the SARS-CoV genome and the amino acid sequences of its open reading frames. Methods are described for using these molecules to detect SARS-CoV and detect infections. Immune stimulatory compositions are also provided, along with methods for their use.
presentation on the molecular biology of Coronaviruses which include taxonomy, history of the viruses, various proteins present in virus, their structure, importance, roles, the life cycle of virus, infection process, the process of disease development, the pathogenicity of virus, replication and translation process in coronaviruses, possible sites for vaccine development, available treatments, cures and drugs, and various studies regarding coronaviruses infection and cures.
Creative Biolabs has extensive experience in coronavirus research. Provide comprehensive high-quality coronavirus (SARS-CoV-2, SARS-CoV, MERS-CoV, etc.) related services and products.
This slide provides a brief introduction to SARS-CoV-2. If you need more knowledge, products and services related to SARS-CoV-2, please follow us.
The document is a United States patent describing methods for producing recombinant coronavirus particles. Specifically, it describes developing a helper cell that contains: 1) a coronavirus permissive cell; 2) a coronavirus replicon RNA containing a heterologous sequence and packaging signal but lacking a structural protein gene; and 3) a separate helper RNA encoding the missing structural protein. When these components are expressed in the cell, coronavirus particles are assembled that contain the heterologous RNA sequence but cannot replicate without the helper RNA. The patent claims compositions and methods for making and using these viral particles to deliver heterologous genes.
This document summarizes research on plant viral suppressors of RNA silencing. It begins by providing background on how RNA silencing was discovered as an antiviral defense mechanism in plants. Most plant viruses encode suppressor proteins that interfere with this silencing pathway. The review then describes the major functional assays that are used to identify these viral suppressor proteins, including transient expression assays using Agrobacterium infiltration and assays to test whether viral infection or expression of a candidate protein can reverse an established silenced phenotype. It focuses on the currently known viral suppressors from different plant virus families and the steps in the RNA silencing pathway that they target.
SARS-CoV-2 was first identified in Wuhan, China in late 2019 and has since spread to over 200 countries. It belongs to the coronavirus family and causes the disease COVID-19. The virus enters cells through the ACE2 receptor and replicates its RNA and proteins before being released to infect other cells. Potential treatment strategies include using existing antiviral drugs, developing specific drugs that target the virus genome or proteins, and enhancing immune responses. Ongoing research focuses on antibody development, diagnostic assays, vaccines, and other medical countermeasures to treat and prevent the spread of SARS-CoV-2.
This study aimed to optimize norovirus GI genotyping primers and apply them to characterize norovirus GI diversity in clinical and environmental samples from South Africa. Five norovirus GI genotypes were found circulating between 2015-2016, with GI.4 being the most prevalent in 63.2% of samples. The primers were optimized to improve genotyping of viruses from sewage samples. National and regional strain clusters were identified, adding to understanding of norovirus genetics and transmission globally.
This document provides information on performance evaluation methods for forensic specialists. It discusses 12 common methods: management by objectives, critical incident method, behaviorally anchored rating scales, behavioral observation scales, 360 degree appraisal, and checklist and weighted checklist method. For each method, it provides a definition and overview, as well as advantages and disadvantages. The goal is to help choose effective approaches for evaluating a forensic specialist's job performance.
Canyon Creek Summer Camp is owned and operated by Daryl Moss, Jeff Robinson, and Nathalie Moss who have over 60 years of combined camp experience. The camp is located on 81 acres in the Angeles National Forest near Los Angeles and features many athletic facilities and activities for campers to enjoy. A typical day at Canyon Creek Summer Camp includes team building activities, sports, water activities at a nearby lake, evening programs, and campfires to help campers build confidence, independence, and lifelong friendships.
The document proposes introducing a new transportation option called the RYNO bike. It would provide an alternative to automobiles for individual travel over short distances. The RYNO bike uses motion sensors and body mechanics to steer and costs about a penny per mile to operate, much cheaper than driving. Introducing this new option could change transportation patterns and business structures. The proposal includes pricing the RYNO bike at $5,295 and budgeting $500,000 for initial advertising to build awareness of the new product.
Heartburn is a burning sensation that occurs in the lower chest when the stomach acid backs up into the esophagus (food pipe). Heartburn is not a serious health concern and can be easily controlled by simple lifestyle changes.
L'Oreal Paris is the world's largest cosmetics brand, established in 1909 in France. It has 2000 products across 500 brands sold in 150 countries. Cosmetics account for 81% of its revenues. In India, the cosmetics industry is worth $950 million and growing at 15-20% annually, projected to reach $2.68 billion by 2020. L'Oreal Paris offers hair, makeup, and skincare products in India across brands like Revitalift, Youth Code, and Total Repair, priced between $1-10. Its target segment is urban, beauty-conscious, multi-tasking new generation women.
Este documento describe las cinco fases principales de un proyecto. Estas incluyen la fase de planificación, la fase de ejecución, la fase de entrega o puesta en marcha, la fase de iniciación y la fase de control. La fase de planificación establece cómo satisfacer las restricciones del proyecto. La fase de ejecución implica realizar las tareas técnicas. La fase de entrega culmina el proyecto y comprueba que funcione según lo especificado.
Cosmic Adventure 3.9-10 World of Finite Light SpeedStephen Kwong
The document discusses how light acts as the carrier of images, allowing humans to see objects. It explains that light travels at a very fast speed of 300 million meters per second, but that there is still a delay in the delivery of images due to the time it takes light to travel from the object to the eyes. This delay means that what we see is actually from the past - the more distant the object, the longer ago the light was emitted. For example, light from the moon takes 1.28 seconds to reach Earth, while light from stars billions of light years away was emitted billions of years in the past. Due to this finite speed of light, the universe can be thought of as consisting of layers of different time periods
This document provides resources and templates for evaluating the job performance of a gaming manager, including:
- Links to free ebooks and forms for performance appraisals on a website.
- Sections of a performance evaluation form covering planning and results, performance factors, employee strengths, areas for improvement, and signatures.
- Example phrases for evaluating a gaming manager's attitude, creativity/innovation, and decision-making in performance reviews.
Haiku Deck is a presentation platform that allows users to create Haiku-style slideshows. The document encourages the reader to get started creating their own Haiku Deck presentation on SlideShare by providing a link to do so. It aims to inspire the reader to try out Haiku Deck's unique presentation style.
This document discusses the scope and challenges of LiFi (Light Fidelity) technology for wireless data communication. LiFi uses visible light communication through LED lights to transmit data, providing higher speeds than WiFi. It has advantages like using the light spectrum, but challenges include not being able to transmit through walls. Researchers are working to advance LiFi technology by developing micro-LEDs that are smaller and faster than normal LEDs to increase data transfer speeds. LiFi could enable new applications if these challenges are addressed.
1. The document provides an exam for a third year secondary student in English as a foreign language. It covers language functions, vocabulary, structure, reading comprehension, and a novel study.
2. The reading comprehension passage discusses the impacts of meteorites on Earth, including a mass extinction event 65 million years ago that wiped out the dinosaurs.
3. The novel questions reference details about the main characters and their interactions in The Prisoner of Zenda by Anthony Hope.
Kathy Dungy is seeking a position that utilizes her diverse education and extensive experience in nursing, education, and sales. She has over 25 years of experience in various nursing roles including case management, disease management, and education. She also has experience in real estate sales and as a substitute teacher and school nurse. Her resume details her education credentials and licenses, achievements, skills, and work history in multiple healthcare, education, and sales roles.
The document lists apartments and rooms for rent in several towns along the Croatian coast near Dubrovnik, including Srebreno, Plat, Mlini, Slano, Štikovica, Cavtat, Brsečine, Orašac, Zaton Mali, and Mlini. It provides links to the rental listings on viaCroatia.com for properties in these towns.
Structure-Based Stabilization of HIV-1 gp120 Enhances Humoral Immune Response...Morteza Loghmani
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immunological impairment allows infectious agents such as viruses, bacteria, fungi and parasites
to invade the body and propagate unchecked. In addition, the incidence of certain cancers
dramatically increases in these patients because of faulty immunosurveillance. AIDS is a serious
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RNA genome and the RNA-dependent-DNA-polymerase termed reverse transcriptase. Members
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sarcomas in humans and animals. The structure and replication mechanism of HIV is very
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in vivo during initial stages of infection and can be transmitted during sexual activity. The HIV
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(glycoprotein) or p (protein) followed by a number indicating the approximate molecular weight
in kilodaltons. The lipid bilayer contains gp120 and gp41. These two proteins are proteolytic
products of the precursor gp160. The gp41 anchors gp120 in the bilayer. The protein gp120 is
routinely used as a diagnostic marker for HIV in Western Blot Analysis. More recently other
viral gp proteins are also included in the test. Beneath the bilayer is a capsid consisting of p17
and p18. Within this shell is the viral core. The walls of the core consist of p 24 and p25. Within
the core are two identical RNA molecules, 9800 nucleotides in length. Hydrogen bonded to each
viral RNA is a cellular tRNA molecule. The viral RNA is coated by tightly bound molecules of p
7 and p 9 . The core also contains approximately 50 molecules of reverse transcriptase. There are
several other viral proteins whose precise functions are not fully understood. The virus can be
grown in tissue culture for diagnostic and research purposes. Several of the viral proteins have
been cloned and generated in relatively large quantities. An individual can receive an inoculum
of HIV through an abrasion in a mucosal surface (e.g., genital and rectal walls), a blood
transfusion, or by intravenous injection with a contaminated needle. Virus or virally infected
cells are found in body fluids such as semen and blood. The most important target for the virus is
hematopoietic cells such as bone marrow derived monocytes, myelocytes and lymphocytes.
Infection of im.
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1. Freeze-Fracture of Electron Microscopy of HIV-1 p24 capsid protein interaction with the
Viral Envelope.
Eric R. Newman
Project Summary
The goal of this research is to further understand how HIV incorporates/attaches
the host cell's lipid membrane as a viral envelope to its RNA enveloping capsid. HIV and
other similar enveloped virus’s biggest competitive advantage is their ability to evade
the host's immune system by hiding behind host cell's membrane complete with host
cell recognition proteins. Research into how this viral envelope recognizes and attaches
to non-infected cells and the mechanism of budding through hijacking of the ESCRT
complex has been extensive. However, research into the actual attachment of the viral
envelope to the capsid is relatively new as it is not a conventional target for treatment.
The goal of this research is to visualize the underlying structure between the viral
envelope and capsid by freeze-fracture electron microscopy to further elucidate the
attachment of the viral envelope to the capsid. Of specific interest is the C-terminal
region of the CA capsid protein that has been shown in previous studies to bind to
lipids. Understanding of this binding event could lead to methods to disrupt this process,
thereby inhibiting infection by HIV and other enveloped viruses.
Specific Aims
The long term goal of this research is to further understand the mechanism of
HIV infection in human cells. Knowledge of how the viral envelope attaches/remains
secure to the capsid enables the possibility of utilizing this complex as a potential drug
or treatment target to prevent and inhibit HIV infection through stripping the virus of its
ability to hide from the host immune system. Without an attached viral envelope the HIV
capsids would be vulnerable to the traditional immune response pathways.
The specific aim of the proposed research is to directly visualize the attached
face of the HIV capsid and the corresponding bottom layer of the viral envelope for
qualitative and visual analysis of the connective elements between the two.
This aim will be carried out through arresting viral bud release by introducing a
premature stop codon into the Gag protein coding for the p6 domain of Pr55Gag,
Freeze-fracture coupled electron microscopy will then be employed to visualize the
arrested and forming viral buds in- and cross-plane with the fractured membrane for a
qualitative look at the capsid/envelope interaction.
2. Background and Significance
Human immunodeficiency virus (HIV) (Figure 1.) is an enveloped lentivirus
responsible for acquired immunodeficiency syndrome (AIDS). AIDS is a syndrome
designated by a low CD4+ T cell count and/or specific diseases and infections
correlated to HIV infection. The HIV virion is spherical with a ~120nm diameter, it is
composed of the viral envelope (phospholipid bi-layer) with its associated membrane
proteins (host and viral) and a protein capsid within which two copies of HIV ssRNA are
located. The capsid is conical and made up of an estimated 2000 copies of capsid
protein monomer. HIV is particularly difficult to treat due to its ability to infect non
dividing cells, genetic variability, and acquisition of host cell membrane during its
budding process. The characteristic of HIV as an enveloped virus is likely the largest
challenge faced by the host immune system in recognizing and eliminating it. As HIV
completes its replication cycle it forms a viral capsid containing two copies of the HIV
ssRNA genome (Moss, 2013). Through the production of proteins coded for by the pro-
viral genetic material integrated into the host cell genome and hijacking of native
proteins, this capsid begins to bud through the host cell membrane. The viral bud
releases from the host cell enveloped in the host’s cell membrane with its associated
proteins (Figure 1.). The integration of the hosts membrane bound proteins help the
virion avoid detection by the immune response. Before budding, bud sites are
proliferated with glycoprotein 41 (gp41) and glycoprotein 120 (gp120) proteins
responsible for the mature virions ability to bind and fuse to a host cell. HIV’s genetic
variability is a product of its rate of replication ~1010 virions daily, mutation rate of 3 x10-5
per nucleotide base pair per replication cycle and the genetic recombination ability
imparted to it through its reverse transcriptase-mediated into the host’s genetic material
(Moss, 2013). This
rate of mutation helps
keep the few elements
that might be
recognized by a host’s
immune system (e.g.,
gp120 and any
degraded protein
fragments picked up
by MHC class 1
molecules) too
variable to elicit any
effective immune
response. The same
challenges the
immune system faces
in targeting HIV are
faced by any
attempted medical
intervention. Though
there are conserved
regions within the
Figure 1. Mature HIV-1 Virion
Used without permission (http://www.scistyle.com/).
3. virus’s proteins and genome, much of it is variable and changes rapidly making a
vaccine unattainable so far, and anti-viral modification only moderately effective. Issues
such as these put constraints on what can be used as viable treatment targets and force
modern science to look at alternate non-conventional approaches to treat HIV.
Modern treatment of HIV consists of five antiviral drug classes. There are
currently 20 approved antiretroviral drugs with the majority being reverse-transcriptase
inhibitors and retroviral protease inhibitors (avert.org). The the most recent classes
consist of fusion of entry inhibitors and integrase inhibitors. The most recent two classes
have had the most success in maintaining virulency against HIV due to the conserved
nature of their targets, HIV's membrane bound glycoproteins and integrase. A issue
with current treatment is HIV's development of resistance to retroviral treatment leading
to a "first line" and "second line" strategy of therapy approach consisting of multiple
waves of a combination of drugs (avert.org).
Global funding in response to HIV/AIDS ranging from care & treatment through
prevention to research is estimated to be $31.7 Billion dollars for 2016 by The Henry J.
Kaiser Family Foundation (Figure 2.). The NIH reported funding for HIV/AIDS research
in the U.S. alone at $3 billion in 2014. According to the World Health Organization
(WHO) there are an estimate 35 million people currently infected by the HIV virus, with
2.1 million new infections during 2013, and HIV is the leading cause of infectious death
Figure 2. Proposed U.S. Federal funding request for HIV/Aids by category.
4. worldwide. Numbers
such as these
emphasize the demand
and importance in
furthering HIV research.
Synthesis of the
capsid protein is carried
out through a
polyprotein precursor the
group-specific antigen
protein (Gag) (Figure 3.)
that is cleaved by HIV
protease into capsid
protein, nucleocapsid,
mature matrix and Gag
proteins. Within the Gag protein
there is a proline rich 6-kDa domain
designated p6 located at the
proteins C-terminal (Figure 4.). This
p6 domain was shown to be integral
to viral bud release from the cell
membrane (Huang, Et al. 1995).
The p6 region consist of several
highly conserved domains implying
function. A nonsense mutation
consisting of a premature stop
codon at the very beginning of the
p6 domain reduced viral bud
release 20 fold (Huang, Et al. 1995).
The long term goal of the
proposed research is to exploit the
viral coat/capsid complex as a
treatment target. Recent
experiments have shown the C-
Terminal domain of the capsid
protein to have lipid binding
properties through fluorescence and
FTIR measurements and it is
believed that this is effect plays a
part in the conjoining of the capsid
and viral envelope (Figure
5.)(Barrera, Et al. 2006).
Understanding of capsid and viral
envelope functional morphology
through imaging of the capsid
Figure 3. Organization of the HIV-1 domains Capsid (CA),
Matrix (MA), Nucleocapsid (NC), protease p1 & p6 (PR),
integrase (IN) and RT domains of the gag and pol reading
frames. Used without permission (Huang, Et al. 1995).
Figure 4. Assembled HIV-1 Capsid composed of
~2000 monomeric copies (Left), Capsid protein
with N-terminal domain & C-Terminal domain of
interest (Top Right), Capsid Pentamer with NTD
displayed in blue (Middle Right), Capsid
Hexamer with NTD displayed in green (Bottom
Right). Used without permission (Deshmukh, Et
al. 2013).
5. protein's interaction with the fore mentioned envelope is intended to be a building block
to further more quantitative research into the strength and perturbations of this
attachment. The viral envelope is one of HIV’s greatest strengths allowing it to hide from
host immune response and an essential component in continuing the HIV life cycle. If
the viral coat could be disrupted or stripped from the HIV capsid it would not cure a
prevailing infection
but could be utilized
much as anti-virals
are today, as an
inhibitor to current
infections and
prevention against
possible infection by
at risk populations.
Disruption or removal
of the viral envelope
would shut down one
of HIV’s two
pathways to continue
its infection of new
cells and the only
pathway to infection
in a new host.
Without an intact viral
envelope the HIV
virion faces multiple
issues. First, without
the glycoproteins
located on and within
the viral envelope
HIV cannot recognize
and bind to the CD4
receptors and CCR5
& CXCR4 co-receptors on the host cell preventing fusion and infection with the inserted
viral genetic material (Figure 3.). Second, without the host proteins associated the
stolen lipid membrane the virion cannot effectively hide from the host’s immune
response leaving it vulnerable.
Freeze-etching is a subset of Freeze-fracturing that employs sublimation after
fracturing, both are techniques employed in tandem with electron microscopy. Electron
microscopy sample chambers are kept at low pressure to avoid electron scattering by
gasses which causes problems with biological samples. Freeze-fracturing freezes the
water within and around a biological sample at high pressure to from amorphous ice an
isochoric process. Fracturing of the frozen sample is conventionally done with a cold
razor blade. The fracture plane commonly splits the inner and outer lipid bilayer but can
also create a fracture plane splitting the lipid bilayer from the cell or split a plane directly
through a cell (Figure 6). When this is done transmembrane proteins are visible either
Figure 5. Crystal structure of monomeric Capsid protein C- terminal
domain with tyrosine & tryptophan residues in stick form. Regions
with predicted lipid membrane affinity are in dark gray including the
asterisk indicated alpha-helix. Used without permission from
(Barrera, Et al. 2006).
6. directly within a separated bi/monolayer or through the void they occupied. Once
sample fixation is achieved an additional step designated etching can be done where
more details of the fractured sample can be visualized if the surrounding ice is removed
through sublimation to maintain sample structure. This etching as done at low
temperature and high pressure to minimize ice crystal formation and remnant gas
deposition both of which contaminate the sample (Severs, 2007). Once the sample has
been prepared it is coated under an angle (~2-8o with rotation for small structure
resolution) with a thin film of heavy metal (generally platinum ~7nm) to create
shadowing for contrast and subsequently a layer of carbon (~15nm) at a 90o angle to
the fracture plane to stabilize the thin metal film. With a simultaneous evaporation of
platinum and carbon monoclonal steps of 11Å have been resolved (Bradley, 1959)
however more common platinum followed by carbon layering routinely have a resolution
of 2.5nm opposed to the 11Å resolution expected out of the methods described later.
Following sample shadowing and coating the remaining biological material is removed
using SDS or acid digestion after which the sample is ready for analysis (Servers,
2007).
Electron microscopy is a form of microscopy that uses an electron beam to
visualize a sample. Due to the extremely short wavelengths of electrons the resolving
power is much higher than that of conventional light microscopy. Transmission electron
Figure 6. (Top) Examples of different fracture planes obtained through Freeze-Fracture.
Exoplasmic Surface (ES), Plasmatic Fracture Face (PF), Exoplasmatic Fracture Face (EF),
Cytoplasm (Cyt). (Bottom) Freeze-Fracture with a PF and integrated proteins.
Used without permission (Source: http://www.leica-microsystems.com/science-lab/brief-
introduction-to-freeze-fracture-and-etching/)
7. microscopy (TEM) has a reported 50 pm resolution and can reach magnification up to
10,000,000x (Ernie, Et al. 2009). TEM utilizes an accelerated electron beam focused by
electromagnetic and electrostatic lenses into a focused beam that gets scattered as it
passes through the sample and the beam is subsequently used to expose a
photographic plate. (Ernie, Et al. 2009).
Altogether the proposed research is viable due to the following reasons. First,
bud arrestment fixing the subject to be studied and increasing the density per area of
buds approximately 20-fold will provide a large amount of sample to be studied (Huang,
et al. 1995). The mutagenesis to achieve this increased bud density has been
performed and verified by previous research (Huang, et al. 1995). Second, the C-
terminal region of the capsid protein has displayed lipid binding properties that imply
some sort of interaction with the lipid bi-layer viral envelope (Figure 5.)(Barrera, et al.
2006). Third, the long term goal of viral envelope degradation/removal would prevent
viral bud infection based on the gp120 protein function and viral envelope location and
gp40 location and function within the viral envelope, nonetheless the speculative
vulnerability of uncoated HIV virion to a host immune response (Figure 1.)(Maartens, et
al. 2014). Fourth, freeze-fracture has been demonstrated to provide fracture planes
through and around inter-membrane and membrane bound vesicles ~40 nm smaller
than the HIV virion (Zampighi, et al. 1999). Fifth, with freeze-fracture electron
microscopy's magnification and a resolving power of 11Å we expect to be able to
resolve the most likely culprit of capsid protein viral envelope interaction the C-terminal
alpha helix due to the 12Å diameter of alpha helices and the variable length a helix or
other subunit could protrude into the viral envelope. We hope to elucidate the
membrane bound and intermembrane proteins within, through and into the viral
envelope (Figure 5)(Ernie, et al. 2009). Last, there is a distinct need and available
funding for further information on HIV especially with pathways leading to conventional
and nonconventional treatment targets (Figure 2).
Research Design and Methods
First, arresting viral bud release will be achieved through site directed
mutagenesis followed by transfection. Viral bud visualization is challenging due to
multiple characteristics of the virion. The challenging characteristics of viral bud
visulization constituting of the viral bud size and migratory. Through site directed
mutagenesis a premature stop codon will be added right after the start codon of the p6
region of the HIV Gag protein (Figure 3.). The p6 region plays a crucial role in the
release of the HIV virion from the infected cell membrane once the viral capsid is fully
formed and disrupting it has been shown to arrest virion release from the host
membrane but still allow virion formation. Using a 1.2-kbp SphI-BalI fragment
(nucleotides 1442-2619) from a full length infectious clone of pNL4-3, the most widely
used vector for in vitro manipulation of the HIV proviral sequences. The 1.2-kbp SphI-
BalI fragment will then be sub-cloned into an M13mp18 single stranded DNA plasmid
and directed-oligonucleotide mutagenesis will be performed as proscribed by (Kunkle,
et. al., 1987). A 473-bp of ApaI-PpuMI fragment (nucleotides 2006-2483) will carry a
premature stop codon mutation in the (PTAP-, residues 7-10) region of the p6 domain of
8. the HIV Pr55Gag protein (Huang, et. al., 1995). This premature stop codon removing
the highly conserved PTAP- motif within the p6 region was shown to inhibit viral bud
release as designated in a 20 fold decrease in viral reverse transcriptase activity
(Huang. 1995). Arresting bud release will give a higher density of attached viral buds for
freeze fracture and subsequent electron microscopy imaging. Due to the small size of
retrovirus particles ~100nm and the inconsistent fracture planes of the freeze-fracture
technique an increased particle density will correlate to an increase in viral buds
fractured along a c-terminal plane while also being visualized in the subsequent electron
microscopy. Following the site directed mutagenesis the ApaI-PpuMI fragment will be
DNA sequenced as a positive control to verify the directed mutagenesis took place.
Two separate cell lines will be studied during the visualization the HIV capsid
stripped of either one or both of the lipid layers constituting its viral envelope in an
attempt to obtain different fracture planes during freeze-fracturing. Cell cultures
containing HeLa 10-15µm and CEM(12D-7) 5-10µm cells will be prepared and as
described in (Freed, et al., 1994). CEM(12d-7) cells are of the T-cell line native targets
for HIV. However, the HeLa cell line has been transfected to express Cluster
differentiation complex 4 (CD4) to allow infection by HIV. Calcium phosphate
precipitation and DEAE-dextran transfection will be used to transfect the HeLa and
CEM(12D-7) cells respectively, both methods described in (Freed, et al., 1994). The
CEM(12D-7) cell line will then be infected with equal amounts of mutated HIV after
normalization for reverse transcriptase activity as described by (Freed, Orenstein, et al.,
1994). Reverse Transcriptase assays for the CEM(12D-7) cell line will performed as
described by (Willey, et al., 1998) and (Freed, Orenstein, et al., 1994) for the HeLa cell
line as controls to determine the successful retardation of viral bud release by the
mutated HIV strain and HeLa cells. The afore mentioned normalization for RT activity is
executed to maintain a standard activity level for the subsequent RT assay to verify
virion release inhibition.
Forty-eight hours post infection, infected cells will be harvested and suspended in
distilled water to a resting culture at 4o-6o C with the sample being utilized before 12
hours has passed to minimize sample degradation. Obtaining and freezing of the HIV
sample by liquid nitrogen at -160oC will be executed as described by (Gross, Kuebler.,
1978). Following sample preparation, freeze-etching, platinum-carbon evaporation,
platinum-carbon shadow coating, and carbon backing will all be executed as described
in (Gross, Kuebler. 1978). Electron microscopy and image processing will also be done
using the procedure as designated by (Gross, Kuebler, 1978). Freeze fracturing will be
done on a modified high vacuum Balzers BA 350 U UHV-Freeze-Fracture apparatus
operating at ~10-9 torr and -196oC as opposed to the conventional ~10-6 torr and -100oC
for improved topographic resolution. The ultrahigh vacuum (UHV) technique was shown
to lessen structural distortion and contamination of the fracture plane with condensed
gasses hiding and create structures. Lowering the temperature during freeze-fracture
reduces distortion of fractured structures, damage caused during radiation heating and
surface diffusion by condensing metal atoms. The low vacuum helps alleviate the one
non-temperature dependent aspect of freeze fracturing, contamination of the sample by
gas molecules 1000-fold (Gross, Bas. 1978). After coating samples will be imaged with
a Siemens 102 electron microscope coupled with an anticontamination device with
9. pictures taken on electron image plates at all followed by detailed image analysis
encompassed in methods proscribed by (Gross, Kuebler., 1978).
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Structure and Dynamics of Full-Length HIV-1 Capsid Protein in Solution. Journal of the
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