Lambda Phage Replication Cycle

Lambda phage morphology:

The head has 20 faces. A three-dimensional image with 20 faces is called an icosahedron. The head is made of proteins of various types and contains 46,500 bp long genomic DNA (g). Phage λ contains circular double-stranded DNA approximately 17 µm in length packaged in the protein head of the capsid. The head is 55 nm in diameter and consists of 300 to 600 37,500 Dalton capsomeres (subunits).

Capsomeres are arranged in groups of 5 and 6 subunits, that is, pentamers and hexamers. The head is attached to a 180 µm long non-contractile tail via a connector. The queue consists of 35 stacked disks. It ends in a fibre. There is a hole in the capsid through which this narrow part of the neck passes which expands into a bulge-like structure on the inside. The tail has a thin tail fibre (25 nm long) at its end that recognizes hosts. Also, the tail consists of about 35 stacked discs or rings. Unlike the T-even phage, it is a simple structure devoid of a tail sheath.

Bacteriophage A belongs to the Siphoviridae family of Group I (dsDNA viruses). Lambda Phage Replication Cycle is an E. coli K12 virus that, after entering the host cell, does not normally kill it, despite being capable of destroying the host. Therefore, it carries out its life cycle in two different ways, one as a virulent virus and the second as non-virulent. The virulent phase is called the lytic cycle and the non-virulent as temperate or lysogenic, and the respective viruses as virulent phage and temperate phage, respectively. The other temperate lambdoid phages are 21, Ø80, Ø81, 424, 434, etc.

DNA and gene organization of lambda phage:

Lambda DNA is a linear, double-stranded duplex approximately 17 µm in length. It consists of 48,514 base pairs of known sequences. Both ends of the 5′ terminus consist of 12 bases that extend beyond the nucleotide of the 3′ terminus. This results in a single-stranded complementary region commonly called sticky ends. The sticky ends form base pairs and can easily circularize.

Consequently, a circular DNA with two single-strand breaks is formed. The double-stranded region formed after base pairing of complementary nucleotides is designated COS. The 12 nucleotides at the sticky ends and the process of circularization. Circularization events occur after injection of phage DNA into the E.coli cell where the bacterial enzyme, ie, E.coli DNA ligase, converts the molecule into a covalently sealed circle.

Lambda phage life cycles:

Following adsorption to the host cell’s lamb receptor, lambda gDNA is injected through the tail, which forms a hollow tube through which the DNA passes into the cell. The phage λ goes through two life cycles, the lytic cycle and the lysogenic cycle after injecting its DNA into the E.coli cell. In the lytic cycle, phage genes are expressed and DNA replicates, resulting in the production of various phage particles. The lytic cycle ends with the lysis of E. coli cells and the release of phage particles. This lytic cycle is virulent or moderate in which the phage multiplies into several particles. Furthermore, the lysogenic cycle results in the integration of the phage DNA with the bacterial chromosome and becomes part of the host DNA.

It replicates along with the bacterial chromosome and is inherited in the progeny. The phage DNA integrated with the bacterial chromosome is called a prophage. The prophage is not virulent and is called a moderate phage. Bacteria containing prophage are called lysogenic bacteria and the prophage stage of viruses as lysogenic viruses. After treatment of lysogenic bacteria with ultraviolet light. X-ray or mitomycin, the prophage can separate from the bacterial chromosomes and enter the lytic cycle. This process is known as induction.

Genetic map of phage lambda:

The notable feature of the map is the grouping of genes according to their functions. For example, the head and tail synthesis, replication, and recombination genes are arranged in four distinct groups. These genes can also be grouped into three main operons, viz. right operon, left operon, and immunity operon. The right operon is involved in the vegetative function of the phage, e.g. head synthesis, tail synthesis, and leading lytic cycle in DNA replication.

The left operon is associated with integration and recombination events of the lysogenic cycle. The products of the immunity operon interact with the DNA and decide whether the phage will start the lytic cycle or the lysogenic cycle. Singer et al (1977) have given the nucleotide sequence of ØX174. The genetic map of bacteriophages has been provided by Echols and Murialdo (1978).

(i) Head Synthesis Genes:

On the far left of the phage genome, the major genes, viz. A, W, B, C, D, E are located which are associated with the maturation of phage DNA and head proteins.

(ii) Tail Synthesis Genes:

The F, Z, U, V, G, H, M, L, K, I, J genes are clustered right in the head genes and code for the tail proteins.

(iii) Cleavage and integration genes:

The xis gene encodes the protein that cleaves phage DNA from bacterial chromosomes, and the int-encoded protein is involved in the integration of phage DNA into the bacterial chromosome.

(iv) Recombination:

The two genes int and xis code att P for site-specific recombination. The three red genes code for three proteins at a normal frequency for general recombination. The red is encoded for exonuclease, the red B for beta protein, and the red V for gamma protein. The gamma protein inhibits exonuclease V.

(v) Positive regulation gene:

The N and R genes are the positive regulation genes. The proteins encoded by these genes increase the transcription rate of other genes. The protein encoded by the N gene induces the transcription of the cell, Q, P, A, red, gam, xis, and int, while the protein encoded by the Q gene stimulates the transcription of the head, tail, and lysis genes. The N and Q genes are also required for plaque formation, in the absence of which the number of phage particles would be lower but not zero.

(vi) Negative Regulation Genes:

The cl gene acts as a repressor, and its product maintains the prophage in the lysogenic form in the bacterial host. In addition, cll and cIII help the d gene in lysogeny. Cro-encoded proteins bind to PL and PR and reduce the expression of cl, N, red, and xis genes. Interactions between the cro-encoded Q proteins and the phage repressor occur in the host cell and the result decides the functioning of the lytic or lysogenic cycle. The choice between lysogeny and lysis was discussed in the previous section.

(vii) DNA Synthesis Genes:

The two genes O and P are involved in the synthesis of phage DNA. The origin of DNA replication lies within the coding sequence of the Q gene, which encodes a protein for the initiation of DNA replication, and the gene that generates the sticky ends lies adjacent to one of the ends. The function of the N gene is required in the transcriptional process of these genes.

(viii) Lysis genes:

The S and R genes control the lysis of the bacterial cell envelope that occurs at the end of the lytic cycle.

The choice between lytic and lysogenic cycles:

Shortly after genome circularization and the start of transcription, gpcII and gpcIII accumulate. gpcII binds to PRE (promoter for the establishment of a repressor) and stimulates RNA polymerase binding. gpcIII protects gpcll from degradation by host nucleases.

The lambda repressor (GPL) is rapidly synthesized (B), binds to OL and OR, and inhibits mRNA synthesis and the production of gpcII and gpcIII (proteins) (C). The repressor activates the promoter for repressor maintenance (PRM) which induces the c/ gene to be continuously transcribed at a low rate. This process continues continuously and ensures stable lysogeny when established (C).

Over the course of time, the pro also accumulates. It binds to OL and OR, activates the transcriptional repressor gene cl, and represses PRM function (D). The (gpcl) repressor can block cro transcription. Therefore, there is a race between the production of gpcl and gpcro proteins.

HIV Treatment

What is HIV?

HIV is a virus that damages the immune system. Untreated HIV affects and kills CD4 cells, which are a type of immune cell called a T cell. Over time, as HIV kills more CD4 cells, the body is more likely to develop various types of conditions and cancers.

HIV is transmitted through bodily fluids including:

  • blood
  • semen
  • vaginal and rectal fluids
  • breast milk

The virus is not spread through air or water, or by casual contact. Because HIV inserts itself into the DNA of cells, it is a lifelong condition and there is currently no drug that will remove HIV from the body, although many scientists are working to find one. However, with medical care, including a treatment called antiretroviral therapy, it is possible to control HIV and live with the virus for many years. Without treatment, a person with HIV is likely to develop a serious condition called Acquired Immune Deficiency Syndrome, known as AIDS.

At that point, the immune system is too weak to respond successfully against other diseases, infections, and conditions. Without treatment, life expectancy with end-stage AIDS is about 3 years. With antiretroviral therapy, HIV can be well controlled and life expectancy can be about the same as someone who has not contracted HIV. It is estimated that 1.2 million Americans are currently living with HIV. Of those people, 1 in 7 do not know they have the virus. HIV can cause changes throughout the body.

The first symptoms of HIV

The first few weeks after someone gets HIV is called the acute stage of infection. During this time, the virus reproduces rapidly. The person’s immune system responds by producing HIV antibodies, which are proteins that take action to respond against infection. During this stage, some people have no symptoms at first. However, many people experience symptoms in the first month after contracting the virus, but often don’t realize that HIV causes those symptoms. This is because the symptoms of the acute stage can be very similar to those of the flu or other seasonal viruses, such as:

  • can be mild to severe
  • they can come and go
  • can last from a few days to several weeks

Early symptoms of HIV can include:

  • fever
  • shaking chills
  • swollen lymph nodes
  • general aches and pains
  • acne
  • throat pain
  • headache
  • nausea
  • Stomach ache

Because these symptoms are similar to those of common illnesses like the flu, a person with them may not think they need to see a health care provider. And even if they do, his health care provider might suspect he has the flu or mono and may not even consider HIV. Whether a person has symptoms or not, during this period their viral load is very high. Viral load is the amount of HIV found in the bloodstream.

A high viral load means that HIV can be easily passed to another person during this time. The initial symptoms of HIV usually resolve within a few months when the person enters the chronic or clinical latency stage of HIV. This stage can last for many years or even decades with treatment. The symptoms of HIV can vary from person to person.

What are the symptoms of HIV?

After the first month or so, HIV enters the clinical latency stage. This stage can last from a few years to a few decades. Some people do not have any symptoms during this time, while others may have minimal or nonspecific symptoms. A nonspecific symptom is a symptom that does not belong to a specific disease or condition. These nonspecific symptoms may include:

  • headaches and other aches and pains
  • swollen lymph nodes
  • relapsing fevers
  • night sweats
  • fatigue
  • nausea
  • vomiting
  • Diarrhea
  • weightloss
  • skin rash
  • recurrent oral or vaginal yeast infections
  • pneumonia
  • herpes

As in the early stage, HIV is still transferable during this time, even without symptoms, and can be passed on to another person. However, a person will not know they have HIV unless they are tested. If someone has these symptoms and you think they may have been exposed to HIV, it’s important to get tested. Symptoms of HIV at this stage may come and go or may progress rapidly. This progression can be substantially slowed with treatment. With consistent use of this antiretroviral therapy, chronic HIV can last for decades and probably won’t progress to AIDS, if treatment is started early enough.

Causes of HIV

HIV is a variation of a virus that can be transmitted to African chimpanzees. Scientists suspect that the simian immunodeficiency virus (SIV) jumped from chimpanzees to humans when people ate chimpanzee meat containing the virus. Once inside the human population, the virus mutated into what we now know as HIV. This probably happened as early as the 1920s. HIV spread from person to person throughout Africa over several decades. Eventually, the virus migrated to other parts of the world. Scientists first discovered HIV in a human blood sample in 1959. HIV is believed to have existed in the United States since the 1970s but did not begin to affect public awareness until the 1980s.

HIV Treatment Options

Treatment should begin as soon as possible after an HIV diagnosis, regardless of viral load. The main treatment for HIV is antiretroviral therapy, a combination of daily medications that stop the virus from reproducing. This helps protect CD4 cells, keeping the immune system strong enough to take action against the disease.

Antiretroviral therapy helps prevent HIV from progressing to AIDS. It also helps reduce the risk of passing HIV to other people. When HIV treatment is effective, the viral load will be “undetectable.” The person still has HIV, but the virus is not visible in the test results. However, the virus is still in the body. And if that person stops taking antiretroviral therapy, the viral load will rise again and HIV can attack CD4 cells again.

HIV medications

Many antiretroviral therapy drugs are approved to treat HIV. They work to stop HIV from reproducing and destroying CD4 cells, which help the immune system mount a response to infection. This helps reduce the risk of developing HIV-related complications, as well as transmitting the virus to others. These antiretroviral drugs are grouped into six classes:

  • Nucleoside reverse transcriptase inhibitors (NRTIs)
  • non-nucleoside reverse transcriptase inhibitors (NNRTIs)
  • protease inhibitors
  • fusion inhibitors
  • CCR5 antagonists, also known as entry inhibitors
  • Integrase chain transfer inhibitors

Treatment regimens

The US Department of Health and Human Services (HHS) generally recommends an initial three-drug HIV regimen from at least two of these drug classes. This combination helps prevent HIV from developing drug resistance. (Resistance means the drug no longer works to treat the virus.) Many of the antiretroviral drugs are combined with other drugs so that a person with HIV usually takes only one or two pills a day.

A health care provider will help a person with HIV choose a regimen based on the person’s general health and personal circumstances. These medications must be taken every day, exactly as prescribed. If not taken properly, viral resistance can develop and a new regimen may be needed. Blood tests will help determine if the regimen is working to keep your viral load down and increase your CD4 count. If one antiretroviral therapy regimen doesn’t work, the person’s healthcare provider will switch to a different regimen that is more effective.

Side effects and costs

Side effects of antiretroviral therapy vary and can include nausea, headache, and dizziness. These symptoms are usually temporary and go away over time. Serious side effects can include swelling of the mouth and tongue, and liver or kidney damage. If side effects are severe, medications may be adjusted. Antiretroviral therapy costs vary by geographic location and type of insurance coverage. Some pharmaceutical companies have assistance programs to help lower the cost.

What tests are used to diagnose HIV?

Several different tests can be used to diagnose HIV. Health care providers determine which test is best for each person.

  • Antibody/antigen tests

Antibody/antigen tests are the most commonly used. They can show positive results usually within 18 to 45 days after someone initially contracts HIV. These tests check the blood for antibodies and antigens. An antibody is a type of protein that the body makes to respond to an infection. An antigen, on the other hand, is the part of the virus that activates the immune system.

  • Antibody tests

These tests check the blood for antibodies only. Between 23 and 90 days after transmission, most people will develop detectable antibodies to HIV, which can be found in blood or saliva. These tests are done using blood tests or oral swabs, and no preparation is needed. Some tests provide results in 30 minutes or less and can be done in a health care provider’s office or clinic.

Other antibody tests can be done at home:

1. OraQuick HIV test. An oral swab provides results in as little as 20 minutes.
2. Home Access HIV-1 test system. After the person pricks their finger, they send a blood sample to an authorized laboratory. They can remain anonymous and request results the next business day.

If someone suspects they have been exposed to HIV but has a negative home test, they should repeat the test in 3 months. If they have a positive result, they should follow up with their health care provider to confirm it.

  • Nucleic acid test (NAT)

This expensive test is not used for general screening. It is for people who have early symptoms of HIV or have a known risk factor. This test does not look for antibodies; look for the virus itself. It takes 5 to 21 days for HIV to be detectable in the blood. This test is usually accompanied or confirmed by an antibody test. Today, it’s easier than ever to get tested for HIV.

HIV Replication Cycle

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Summary

Inhibition of HIV replication initially focused on viral enzymes, which are exclusively expressed by the virus and are not present in the human cell. The development of reverse transcriptase (RT) inhibitors began with the discovery of the antiretroviral activity of the nucleoside analogue zidovudine in March 1987. Currently, six main classes of antiretroviral drugs are used for the treatment of HIV-infected patients: RT inhibitors, nucleoside and non-nucleoside inhibitors, protease inhibitors, the integrase inhibitors raltegravir, the fusion inhibitor enfuvirtide (T-20), and the 5-chemokine receptor antagonist maraviroc.

A seventh class, the maturation inhibitors, have not yet been approved because their efficacy is affected by HIV-1 polymorphisms that occur naturally in 30-40% of untreated HIV-1 isolates. The use of antiretroviral combination therapy has been shown to be effective in slowing the progression to AIDS and in reconstituting the immune system of people infected with HIV. Over the past 5 years, the introduction of newer antiretrovirals has greatly increased the effectiveness of treatment. However, the development and accumulation of resistance to all classes of antiretroviral drugs remain a major problem. Additional goals will need to be defined to achieve the ultimate goal: the eradication of the virus from the infected human body.

Reverse transcription inhibitors

After the release of the capsid into the cytoplasm, the capsid and nucleocapsid disassemble (mismatch), although the precise mechanism is still unknown. Genomic RNA is associated with viral tRNALys and with several viral proteins such as RT, IN, PR, Vpr, and MA that constitute the reverse transcription complex (RTC). The RTC uses the microtubule system for transport through the cytoplasm. Within the RTC, reverse transcription of viral RNA into DNA occurs by viral RT, although the efficiency of reverse transcription is highly dependent on the presence of all components of the RTC. For example, in the absence of the IN protein, reverse transcription is completely blocked.

RT is an RNA-dependent DNA polymerase that produces double-stranded DNA from single-stranded RNA. This process begins with the synthesis of negatively oriented single-stranded DNA copied from viral RNAs, which is used as a template for subsequent second-strand DNA synthesis. RT is a heteromeric enzyme that comprises a regulatory subunit (p51) and a catalytic subunit (RNase H – p15) that form the p66 molecule. p66 resembles a right hand, where the subdomains are designated fingers, palm, and thumb. The catalytic site is located in the palm and comprises amino acids D185-D186 and D110, a highly conserved motif also in other RTs and polymerases.

It includes the activity of viral ribonuclease H, responsible for the degradation of the template RNA of the DNA/RNA hybrid. Since HIV-1 RT is reported not to maintain sustained replication for more than about 100 to 200 bases, reverse transcription is the replication step with the highest probability of recombination events between the two HIV-RNA strains. 1 in each particle. Like all RNA polymerases, HIV RT has a high error rate when transcribing RNA into DNA, as it has no proofreading ability. This high error rate, in combination with the high rate of recombination, allows mutations to accumulate at a rapid rate, which has important implications for immune escape, development of drug resistance, and tropism switching, among others.

IN inhibitors

Different substances are currently being developed, but only one licensed for clinical use belongs to the so-called chain transfer inhibitors. These drugs bind to IN near the DDE motif in the active site and competitively block IN activity. Proviral DNA cannot be inserted into the host genome and is circularized by cell repair enzymes, irreversibly stopping viral replication.

Raltegravir (RAL, Isentress®, Merck) is a chain transfer inhibitor with potent activity against HIV-1 and HIV-2. RAL is administered orally twice daily, does not require RTV boosting, and is well tolerated. Results from clinical trials indicate that RAL is safe and highly effective in the treatment of antiretroviral treatment-naïve and antiretroviral treatment-experienced patients. Resistance to RAL has been associated with amino acid substitutions in three key positions of the IN protein: Y143R/C, Q148H/R/K or H155H, alone or accompanied by other mutations such as T66I, L74M, E92Q, T97A, E138K+ G140S/ To GY143H, V151I and G163R.

Elvitegravir (EVG, GS-9137, Gilead), a second-chain transfer inhibitor, is in phase III clinical trial. It is also active against HIV-1 and HIV-2. EVG has the advantage of once-daily oral dosing when boosted with RTV. EVG resistance is associated with mutations T66I/A/K, E92Q, E138K, Q146P, S147G, Q148R/H/K, and N155H, which are close to RAL-selected resistance mutations; therefore, cross-resistance for both drugs is expected. Other drugs currently in development are reviewed in Serrao et al.

Maturation inhibitors

Maturation inhibitors are drugs that target one or more cleavage sites within Gag precursor proteins or that inhibit the capsid protein interactions required for core condensation. Bevirimat (PA-457; Myriad Pharmaceuticals) is the first compound in its class, although the drug has not yet been approved by the FDA and the EMEA. Bevirimat is inactive against HIV-2.

HIV-1 mutations conferring resistance to bevirimat were located at the P24/P2 cleavage site (H358Y, L363M/F, A364I/M/V, and A366V/T) and at the P2 peptide (Q369H, V370A/M/ del and T371del), either by increasing the rate of cleavage at the P24/P2 site by viral PR or by interfering with drug binding. Unfortunately, the efficacy of bevirimat therapy is affected by HIV-1 polymorphisms at P2 (amino acids 369–371), which occur naturally in 30–40% of treatment-naïve isolates of HIV-1. In addition, co-evolution of HIV PR and Gag mutations has been observed during PI exposure, and PI treatment failures increase the prevalence of bevirimat resistance and reduce clinical outcomes during bevirimat therapy.

Conclusions

The use of combination antiretroviral therapy has been shown to be effective against progression to AIDS in HIV-infected individuals. Over the past 5 years, the introduction of two new PIs (DRV and TPV) with broad activities against PI-resistant viral strains, the CCR5 antagonist maraviroc, the IN inhibitor RAL, and the second-generation NNRTI ETR, have greatly increased the efficacy of the drug. antiretroviral treatment. Meanwhile, successfully treated HIV infection can be considered a chronic disease rather than a fatal infection.

However, the success of antiretroviral therapy is limited by high costs, the development of viral resistance, and side effects. The eradication of the virus from the infected body by combination antiretroviral therapy or the cure of HIV infection is still not possible. Additional goals will need to be defined to achieve the goal of medical intervention in HIV infection: a global perspective for surviving HIV infection to normal life expectancy.