Bacteriophage lambda (λ) was discovered by Joshua and Esther Lederberg. While mutagenizing strains E. coli using UV, a strain was found to be a lysogen.

• Genome is 48,502 bp dsDNA with 12 nucleotide ssDNA cohesive termini. Contained in capsid.
• Linear dsDNA circularizes due to annealing between 3′ sticky cos sites.
• The capsid has two parts:
  1. Head, composed of B, C, Nu3, D, and E proteins.
  2. Tail, composed of J and H proteins.
• Viral Receptor is the E. coli maltose receptor (product of lamB) on surface of host cell.
• Temperate phage, meaning that it can undergo either lysogeny or lysis.
• Chooses developmental pathway depending on nutrient availability.
• Bacterophage λ has two different life cycles.
  • Lytic, where new phages are synthesized, which then lyse the host and burst from the cell.
  • Lysogenic, where the phage genome integrates into the host genome. It is replicated along with host genome host cell replication. Such a host is called a lysogen.

Int

Pi

tL

N

OL,PL

cI

PRM

OR3

OR2

OR1

PR

Cro

PRE

cII

O

P

tr2

Q

/ /

——–

———–

——

——

——

//

——-

	protein coding gene required for lytic replication
	promoter active during lytic replication
	protein coding gene required for lysogeny
	promoter required for lysogeny
PR	The rightward promoter, transcribed to make Cro & extended by N to make lytic cycle genes O & P. OR3 overlaps PRM.
PL	The leftward promoter, transcribed by Pol to make N. Later extended by N to make the integrase gene for lysogeny.
Hfl	A cellular protease cleaving cII. Function at high [glucose], promoting lysis.
N	N is a viral protein that extends transcription from PR into O and P and from PLinto integrase gene. DNA binding protein and RNApol cofactor, binds DNA (at Nut sites) and transfers onto any oncoming RNApol. Alters the recognition of stop codons, so normal stop codons are ignored and special N stop codons are effective instead.
cII	Made from PR promoter, cII activates PRE to make cI and activates Pi to make integrase. In this way, cII promotes lysogeny. cII outcompetes Cro, unless Hfl protease degrades cII (this only happens when there is high [glucose]. cII is unstable due to succeptability to cellular proteases (especially in healthy cells and cells undergoing the SOS response), slightly stabilised by binding to cIII.
Cro	Made from PR promoter, Cro represses transcription from PRM by binding to OR3. At high concentrations it binds to OR2 & OR1, thereby blocking Pol from binding to PR. Cro is a repressor just like cII, but it binds the operators (like OR3) with the opposite affinity from cI. When Cro binds OR3 it represses transcription of cI gene from PRM. RNAP is already trancribing CRO from PR as well as O and P genes with help from N. At higher levels, CRO also autoregulates itself by binding to OR2 and OR1 and repressing transcription from PR. Transcription inhibitor, binds OR3, OR2 and OR1 (affinity OR3 > OR2 > OR1, ie. prefferentially binds OR3). At low concentrations blocks the R promoter (preventing cI production). At high concentrations downregulates its own production through OR2 and OR1 binding.
cI	Made by activation of PRE promoter through action of cII on PRE, cI is an activator of PRM, recruiting RNAP to OR3 by binding cooperatively to OR1 and OR2. Simultaneously represses PR, occupying RNAP site near OR1. At high [cI], binds to OR3 to block polymerase’s ability to transcribe cI gene, shutting itself off. Transcription inhibitor, binds OR1, OR2 and OR3 (affinity OR1 > OR2 > OR3, ie. prefferentially binds OR1). At low concentrations blocks the RM promoter (preventing cro production). At high concentrations downregulates its own production through OR2 and OR3 binding. Also inhibits transcription from the L promoter. Succeptable to cleavage by RecA* in cells undergoing the SOS response.
cIII	cII binding protein, protects cII from degradation by cellular proteases.
PR & PL	Strong promoters. The other promoters must be activated. PRE is the promoter for repressor establishment.
Q	DNA binding protein and RNApol cofactor, binds DNA (at Qut sites) and transfers onto any oncoming RNApol. Alters the recognition of stop codons, so normal stop codons are ignored and special Q stop codons are effective instead.
xis	excisionase and integrase regulator, manages excision and insertion of phage genome into the host’s genome.
int	integrase, manages insertion of phage genome into the host’s genome. In Conditions of low int concentration there is no effect. If xis is low in concentration and int high the n this leads to the insertion of the phage genome. If xis and int have high (and approximately equal) concentrations this leads to the excision of phage genomes from the host’s genome.
A-F	code for phage head genes.
Z-J	Z-J code for phage tail genes. The order shown here is as found on the genome, reading in a clockwise direction]; structural proteins, self assemble with the phage genome into daughter phage particles.
S, R	Lysis promoters, cause the host cell to undergo lysis at high enough concentrations.
OP	[Shown on diagram as O replication P]; DNA replication promoter, promotes the specific replication of only the phage genome.
SIB	Not a protein, but a vital conserved DNA sequence]; Forms a stable hairpin loop structure in transcribed mRNA. Attracts degradation of mRNA by RNAaseIII.
attp	attP not a protein, but a vital conserved DNA sequence]; point of action of int and xis in insertion and excision of the phage genome into the host’s genome. Corresponding attb found in the host’s genome at the point of insertion.

Lifecycle

The ‘late early’ transcripts continue being written, including xis, int, Q and genes for replication of the lambda genome.
The lambda genome is replicated in preparation for daughter phage production.
Q binds to Qut sites.
Replication from the R’ promoter can now extend to produce mRNA for the lysis and the structural proteins.
Structural proteins and phage genomes self assemble into new phage particles.
Lytic proteins build sufficiently far in concentration to cause cell lysis, and the mature phage particles escape.

[xis and int regulation of insertion and excision]
xis and int are found on the same piece of mRNA so approximately equal concentrations of xis and int proteins are produced. This results (initially) in the excision of any inserted genomes from the host genome.
The mRNA from the L promoter forms a stable secondary structure with a hairpin loop in the sib section of the mRNA. This targets the 5′ end of the mRNA for RNAaseIII degradation, so a lower effective concentration of xis mRNA than int mRNA is found, so higher concentrations of xis than int.
Higher concentrations of xis than int result in no insertion or excision of phage genomes, the evolutionarily favoured action – leaving any pre-insterted phage genomes inserted (so reducing competition) and preventing the insertion of the phage genome into the genome of a doomed host.

The lambda repressor is a dimer also known as the cI protein. It regulates the transcription of the cI protein and the Cro protein. cI and Cro proteins regulate λ life cycle. If cI predominates, the lysogenic cycle will ensure. If Cro proteins predominate, the lytic cycle will ensue. cI dimer binds to OR1, OR2, and OR3 in the order OR1 > OR2 > OR3. Binding of a cI dimer to OR1 enhances binding of a second cI dimer to OR2, an effect called cooperativity. Thus, OR1 and OR2 are almost always simultaneously occupied by cI. However, this does not increase the affinity between cI and OR3, which will be occupied only when the cI concentration is high.

• In the absence of cI proteins, the cro gene may be transcribed.
• In the presence of cI proteins, only the cI gene may be transcribed.
• At high concentration of cI, transcriptions of both genes are repressed.

How does lambda replicate and package viral DNA? Nu1/A (terminase) binds cos sites.

• dsDNA 40kb genome.
• Complex icosohedral head & short tail.
• More completely dominates cell than M13.
• Replication cycle starts; within 6 minutes host cell protein synthesis shut off.
• Viral proteins completely degrade host cell DNA.
• Host nucleotides used for virion genome replication
• Per cell, virion makes 200 copies of viral DNA within 15-20 post-infection.

T7 good model roganism for gene rexpression….dsDNA….simply 55 gene 40kb genome….infect many viral DNAs per cell…isoalte viral mutants

Pulse-Labeling of viral proteins in E. coli

• Add T7 (MOI of 5pfu/cell) at t=0
• Remove 5mL aliquots every 2min
  • Add ³⁵S-methionine for 30 sec (this makes newly synthesized fluoresce)
  • Lyse cells in SDS
  • Subject to polyacrylimide gel electrophoresis
  • Prepare autoradiogram of gel

Steps to Identify Regulatory Mechanism for Gene Expression

There are several possible ways the virus can regulate gene expression.

• Translational or transcriptional control
• Repressors
• RNAP modification
• Viral-specific polymerases

The experiment to determine which mechanism is used goes as follow:

• Identify Class I proteins
• Map location of each Class I gene by mapping RNA for each protein
• Make mutants in each Class I gene
• Analyze the phenotype of each mutant

To identify Class I proteins, proteins in T7-infected cells are pulse-labeled at various time post-infection. Next, the temperature-sesitive mutant experiment is performed. Temperature-sensitive mutants grow at the permissive temperature of 32°C; they do not grow at 39°C. Temperature-sensitive mutants in gene 1 do not express Class II and Class III genes at the non-permissive temperature. The methodology is as follows:

• T7-infect cells are pulse-labeled at various times post-infection.
• Profile of TS-infected cells at non-permissive temperature is compared to the profile of TS-infected cells at permissive temperature.

Are TS1 mutants blocked at the level of transcription? protein profile at non-permissive temperature? aAre ts1 mutants blocked at txnal level? test….infect cells with nhigh MOI at nonpermissive, label with ³H-uridine…extract RNA and hybridize to cloned DNAs of each class of gene….

There is an experiment designed to test this idea:

• Infect cells with high MOI at the nonpermissive temperature.
• Label with ³H-uridine.
• Extract RNA and hybridize to cloned DNAs of each class of gene.

TS1 mutants in gene 1 do not express Class II and Class III genes at the nonpermissive temperature. We design an experiment to see if there is a polymerase encoded in Gene 1 that is necessary to transcribe Class II and Class III genes. The experiment is as follows:

Gel Filtration: An extract of E. coli proteins is poured over a sizing column. Proteins go in and out of beads with different sized holes. Larger proteins elute first; smaller proteins elute later because they get trapped inside the beads. We find a 450kD cellular RNAP and the 98kD viral RNAP. the cellular RNAP has activity initially, but then the viral RNAP replaces it to transcribe the later genes. The conclusion: Gene 1 encodes a T7-specific RNAP responsible for transcribing Class II and Class III genes.

The temporal order of T7 gene expression is in the same order as the genes themselves, from left to right. There is a terminator for E. Coli RNAP before at the beginning of Class II and Class III genes. Class II and Class III promoters have a common DNA sequence different from Class I promoters. Class I promoters are transcribed by the E. Coli’s RNAP. Class II and Class III promoters are transcribed by the T7-encoded RNAP, which recognizes its own promoter sequence.

Summary of T7 Experiments:

• PAGE gel showed three waves of viral proteins.
• Temperature sensitive mutants were developed in Class I genes.
• The phenotype of gene I mutants was that there was no transcription of viral Class II and Class III genes at the non-permissive temperature.
• Extracts from WT infected cells show a viral specific RNAP eluting from an ion exchange column.
• Exctracts from TS1 infected cells at the non-permissive temperature do not show this viral RNAP activity.
• The conclusion is that gene 1 encodes a T7-specific RNAP responsible for transcribing Class II and Class III promoters.
• Class II and Class III promoters have a common sequence different from Class I promoters, which are transcribed by the cell’s RNAP.

This figure shows the activity of the cellular RNA polymerase and the activity of the T7 RNA polymerase as a function of column fractions. How do you think the activity of each of the RNA polymerases was measured? Viral RNAP activity replaces and is greater than cellular RNAP. Activity could be measured by labeling the promoters with different fluorescent genes. The degree of fluorescene would be proportional to RNAP activity.

Intro

Bacteriophage M13 has a 6kb circular ssDNA genome. It is filamentous (as opposed to icosohedral). Special proteins at the tips are involved in assembly, morphogenesis, adsorption and penetration. It infects F⁺ E. coli, but rather than killing host cells it just slows growth. Virions leak out from the cell. Thus, the virus is not very lytic.

Infection

M13 absorbs the tip of the F-pilus and injects its genome into the cytoplasm of the infected cell. Immediately after injecting the viral DNA into the cytoplasm, the circular ssDNA is converted into dsDNA. The dsDNA is transcribed by host RNAP into viral mRNAs. These mRNAs are translated by host cell ribosomes into viral proteins. Proteins then direct replication of viral DNA by host cell enzymes. Progeny virons assemble.

How is dsDNA converted to dsDNA?

• Strand synthesis following infection was blocked by treateing cells with E. coli RNAP inhibitor.
• Do you need pre-existing RNAP for ssDNA -> dsDNA? Find out if new viral gene expression is not needed for M13 genome replication.
  • Infect cells in presence of chloramphenicol (inhibits new protein synthesis).
  • Result: dsDNA is still made.
  • Conclusion: No new protein synthesis required for synthesis of viral dsDNA.
  • Transform naked viral ssDNA into cells.
  • Result: a small amount is converted to dsDNA.
  • Conclusion: capsid proteins not involved.

M13 ssDNA -> dsDNA

These studies revealed that the single stranded viral DNA becomes coated with E. coli Ssb (single-stranded DNA binding protein), except in one region where the sequence forms a hairpin structure. E. coli RNA polymerase can initiate RNA synthesis at this position on the M13 + strand because it is sufficiently double-stranded for RNA polymerase to use as a template. Once the – strand RNA primer is synthesized by RNA polymerase, it is extended around the circle of the + template strand by E. coli DNA polymerase III: Ssb is released as the – strand is synthesized. The RNA primer is then removed by digestion by the 5’ to 3’ exonuclease activity of DNA polymerase I. The primer is replaced by the DNA polymerase activity of DNA Pol I. Finally, the DNA ends are ligated together by DNA Ligase.

M13 DNA Synthesis

We are trying to take the ssDNA, get a capsid around it, and then get it out of the cell. We initially have circular dsDNA. pII knicks the + strand and binds to the end of the + strand. This allows DNAPIII and helicase together duplicate the strand to make another + strand. When pV accumulates late in infection, it binds to ssDNA and replaces the ssDNA binding protein. pVIII binds to the inner membrane. After pV coats the DNA, pVII and PIX binds to the morphogenic sequence. At the same time, pI and pIV compose a secretion apparatus in the membrane. pV interacts with pVIII and the DNA is passed through the secretion apparatus, with pVIII replacing pV. pIII and pVI are added to the tips of the DNA. At the end, there is one strand of DNA with pII, pIII, PVI and pVIII bound to it.

Proteins

• pI comes a secretion apparatus, it is involved in making a pore in the membrane.
• pII first knicks the dsDNA and binds to the end of the + strand. From that, DNAPolIII and helicase replicate the + strand.
• pIII (along with pVI) forms the rounded tip, and is the first protein to interact with E. coli.
• pIV (along with pI) form the secretion appartus, thus making a pore in the membrane. pI and pIV make a pore.
• pV displaces ssDNA binding protein. It interacts with (and is dispaced by) pVIII, thus guiding the virion toward and through the secretion appartus.
• pVI (along with pII) forms the rounded tip, and is one of the first proteins to interact with E. coli.
• pVII (along with pIX) bind to the morphogenic sequence. It interacts with pV and helps initiate phage secretion.
• pVIII lines the inner membrane. It interacts with (and replaces) pV as the phage exits the cell. The secreted virion is surrounded by pVIII rather than pV.
• pIX (along with pVII) binds to the morphogenic sequence. It interacts with pV and helps initiate phage secretion.

Questions

How do you initiate DNA replication from a dsDNA circle?
How do you separate ssDNA circles needed for further DNA replication from those to be packaged?
How do you put a capsid around the DNA and get it out of the cell?
How do you initiate transcription from the dsDNA circle?

• Protein II (pII) nicks the + strand.

How do you separate ssDNA circles needed for further DNA replication from those that are to be packaged?

• pV accumulated and binds displaced _ strand in place of El coli Ssb. = strand bound by pV does not serve as a template for – strand synthesis. M13 + strand replication…describe the cycle in detail.

At the origin of replication, a hairpin structure is formed that allows host cell RNAP to bind. RNAP lays down RNA primers in the. Possible ways for M13 to express genes: host cell zynes, virus encodes enczome packaged in capsid, encodes during lytic cycle

SSDNABindigProtein = SSB

M13 phage display libraries. It is a good template for doing DNA synthesis and mutagenis because of this ss circle and put primer as bridge and 15 mutations in the middle and mutate mutation in promoter or gene like put stop codon in gene.

Steps in phage display:
A peptide introcuced at N-terminus of major coat protein (gene VIII) by PCR mutagenesis
The linear PCR product

A billion porteins with slightly different protein shapes. As a protein peptide, it can bind to different things. By third process of sticking,, washing, eluting, growing…you can amplify specific peptide binding to protein.

Alternatives…mutage gene III not gene VIII. 2500 copies of gene VIII protein per phage and only 5 of gene III prtoein.

It has a long and unusual shape:

The picornavirus family has these general properties:

• Large groups of viruses with common structure, genome organization, and expression
• Genome is a single strand of + sense RNA enclosed in an icosohedral non-enveloped capsid
• Causes a wide range of diseases including poliomyelities, enteitis, hepatitis, and common colds.

The picornavirus family has these 5 genuses, with a popular species of that genus listed next to it:

• Enterovirus (poliovirus)
• Rhinovirus (human rhinovirus 1A)
• Hepatovirus (hepatitis A virus)
• Cardiovirus (encephalomyocarditis virus)
• Aphtovirus (foot and mouth disease virus)

Poliovirus will be examined more closely as a model organism for the picornavirus family. As the first virus to be grown in culture (by Dr. Enders in 1949), it has been heavily studied. There are 2 vaccines used to combat poliovirus:

The course of infection is as follows:

• Like all enteric viruses, it is secreted in feces.
• Fecally contaminated food and water transmit the virus.
• Once the virus is ingested, it infects the gut.
• The primary viremia (first time the virus enters the bloodstream) leads to fever, drowsiness, headache, nausea, and vomiting, followed by recovery within a few days.
• However, in rare cases, poliovirus can invade the CNS and target neurons. Paralysis ensues due to neuronal damage.

There are many ways for the virus to be transmitted. Although usually it results from sewage leaking into a water supply, it can also happen if somebody does not wipe well and then goes swimming in a pool. Since poliovirus can be such a debilitating disease, vaccination is very important. However, it is not necessary to vaccinate everybody. By a phenomenon called herd immunity, where just enough people are treated to block transmission, the disease can be eradicated. Continued vaccination is crucial, though, because lab stocks are maintained (and can be released) and vaccinated patients shed attenuated virus (which can mutate and become pathogenic).

In the absence of any viral proteins, the poliovirus nucleic acid can still produce all proteins needed to make new virion particles. If it had been minus sense, it would need to be transcribed before use as a template for translation.

The 5′ end of the genome is highly structured. Poliovirus contains a very small protein at the 5′ end called VPg. VPg is attached covalently (phosphodiester bond) to the uridine residues at 5′ end of the RNA chain. VPg is present in packaged virion RNA but not on viral RNA that is being translated by cellular ribosomes. Upon infection, a cellular enzyme cleaves VPg from VPg-containing viral RNA. poly(A) is also unusual because it is encoded in the genome, as opposed to being added after transcription. This means it is genetically coded (copied from poly(U) at 5′ end of minus strand during replication) and not added post-transcriptionally.

The CAP structure is crucial for positioning mRNA on ribosome for translation. How does the poliovirus RNA translate? Instead of the ribosome binding to the 5′ CAP, the ribosome binds internally to the 5′ UTR. It is highly structured, with many hairpin loops. There is the VPG, several loops, then the Internal Ribosome Entry Site (IRES) or Ribosome Landing Pad (RLP). At the 3′ end of the IRES is the initiating AUG. There is only one ORF in polio RNA, and no stop codon between protein coding sequences. This indicates the presence of a single long polyprotein. Internal binding selects 9th AUG in poliovirus RNA (same as mRNA). Mature viral and structural proteins are generated by proteolytic cleavage of a long precursor polypeptide (polyprotein). Cleavages occur fast, so it is difficult to detect this large polyprotein. Strucutural proteins on left translated before non-structural proteins on right. Ineffcient. Non-structural proteins like RNA-dependent RNAP 3D^pol not needed in high oconcetrations.

• Viral 2A proteasse functions while still part of polyprotein precursor. Before termination, it auto-catalyzes cleavage of P1 from the growing polypeptide chain.
• Viral 3C protease is on right end of genome and catalyzes generation of individual proteins
• Viral proteases also function to shut down host cell translation and transcription, thus allocating more resources to viral processes.
  • Eukaryotic translation: unwinding beings at 5′ end by binding of the 5′ cap by cellular protein complex (cap-binding protein, CPB, or eIF-4F) which consists of eIF-4A, 4B, and 4E-p220). Poliovirus lacks a CAP, so it doesn’t depend on p220 for it’s function. 5′ UTR secondary structure recognized by cellular p52 and other proteins. Still recruits eIF4a and 4B to unwind secondary structure. Ribosome binds internally. So, cleavage of p220 by viral 2A inhibits cellular protein synthesis but not poliovirus protein synthesis.
  • eIF2 is involved in recruiting tRNA^met-Met to ribosome and positioning the initiator tRNA^met onto AUG. In this energy-dependent process, Gtp bound to eIF2 is hydrolyzed to GDP. Recycling of eIF2 requires exhange of GFP for GTP to start antoher round of recruitment. GEF (GDP exchange factor) catalyzes this. eIF2 recruits met-tRNA to the 40s ribosome and poliovirus proteins block eIF2 recycling. eIF2 phosphorylation prevents guanine nucleotide exhachange, nblocking recruitment of tRNA to reibosome and formotation of 40s complex.
  • Also, trancription is halted. Viral protease 3C is translocated and cleaves transcription factor TBP (TATA-binding protein) and other factors, leading to cellular transcription shut-off.
  • Poliovirus protease 2A shuts off translation by phosphorylated eIF2 and cleaving p220; poliovirus protease 3C shuts off transcription by TBP and TFIIIC.

The fate of newly synthesized plus sense RNA depends on the time after infection. Newly synthesized plus sense RNA has 3 fates:

• Translation (early)
• Replication Template (requires viral replicase)
• New Viral Genomes (late)

The viral replicase, an RNAP, is unable to initiate synthesis from RNA. This is unusual. Like DNAP, it needs a primer. VPg-uridine (VPg attached to uridine) serves as this primer. The viral replicase copies +RNA to make complementary -RNA. This minus strand is the template for many new plus strands. The replicative intermediate (RI) is one minus strand template hydrogen bonded to many nascent plus strand RNAs and vice-versa.

There are 5 miscellaneous topics which are nonetheless very important:

Like togaviruses and flaviviruses, coronaviruses have the following properties:

• + strand ssRNA genomes
• Use insects as secondary hosts
• Wide range and severity of diseases

Coronaviruses have a helical nucleocapsid. They also have an enormous genome (4-5 times larger than that of picornaviruses). While togaviruses use only two mRNAs to synthesize its proteins, coronaviruses use 7. Each coronavirus mRNA encodes for a different protins. Each coronavirus mRNA has the same 3′ sequence (including polyA), but each one begins at a different point on the genomic strand. Only the most 5′ gene is translated from each subgenomic RNA. We will analyze the replication cycle of Mouse Hepatitis Virus (MHV) for further detail:

Like togaviruses, coronaviruses are mainly distributed in the Americas, Africa, and Asia. They prefer topical, hot, and humid climates. Most of the viruses (especially α) use insects/mosquitos/ticks as 2° hosts. They use other animals as reservoirs to maintain virus in nature when 1º is not available.

Like coronaviruses and flaviviruses, togaviruses have the following properties:

• + strand ssRNA genomes
• Use insects as secondary hosts
• Wide range and severity of diseases

There are two classes of togaviruses:

The most important feature of togavirus replication is that is has a dicistronic genome. Two mRNAs are used, one to produce non-structural proteins and another to produce structural proteins. This is important because it is very efficient. Picornaviruses produce one polypeptide, which means that equal amounts of each protein are produced even though unequal amounts are needed. As a result, picornavirus replication is inefficient. Togaviruses, however, regulate synthesis of non-structural and structural proteins by using two mRNAs. In fact, there is 4 times as much subgenomic RNA as genomic RNA in cells infected with togavirus. Two classes of proteins are synthesiszed from different mRNAs, which allows temporal regulation and qualitiative replication. Genomic RNA can do two things: make non-structural proteins (such as replicase) by synthesizing the minus strand, or make structural proteins through synthesis of subgenomic RNA and synthesize positive strand RNA.

A minus strand is made that is complementary to the genomic plus RNA. This minus strand has two initiation sites for replicase: one for non-structural proteins and one for structural proteins. The site for non-structural proteins is at the 3′ end, and if replicase initiates there it will produce the plus RNA encoding non-structural proteins. The site for structural protiens is about mid-way through, and if replicase initiates there then a subgenomic (smaller than genomic) plus RNA will be synthesized. This will encode structural proteins.

Togavirus subgenomic RNA was discovered via the following experiment:

• Cells are infected with togavirus and incubated in labeled uridine
• This labeled uridine is incorporated into all RNA
• Actinomycin D shuts off synthesis of cellular RNA
• As a result, only viral RNAs are labelled
• This RNA is run on a gel, and 3 kinds of togavirus RNA are detected:
  • Genome (plus strand)
  • Negative strand
  • Subgenomic
• Further experiments revealed how the 2 mRNAs could be synthesized at different levels, and that each had only one ORF.

There are five structural proteins: capsid, E1 (composed of E2 and E3), and p62. The capsid protein autolytically cleaves itself from the other structural proteins. The capsid protein assembles with the genomic RNA into a nucleocapsid. E1 and p62 proteins contain a sequence which directs them into the lumen of the endoplasmic reticulum. They move to the Golgi and the trans-Golgi as if they were host cell proteins. They are also modified just like host cell proteins via glycosylation and further cleavage to p62, E2, and E3. Glycoproteins E1, E2, and E3 form a trimer in the cell membrane, which becomes the viral envelope. Incorporation of this trimer into the membrane is very important for the viral infectivity. Cytoplasmic tails of the viral glycoproteins are also required to bind to the nucleocapsid and ensure the membrane forms aorund the new viruses.

Human Immunodeficiency Virus (HIV) is the causative agent of AIDS. It is a retrovirus belonging to the lentivirinae family. HIV reverse-transcribes its RNA to DNA and then back to RNA.

HIV Infection

Leeches and mosquitoes are unable to transmit HIV because the virion is unable to replicate outside of a warm mammalian host. Due to HIV’s specific temperature and environment requirements, it is transferred between mammals.

Cellular requirements for productive HIV-1 replication:

• CD4 surface molecule
• Co-receptor expression
  • CCR5 – the majority of new infections are by viruses that utilize this co-receptor; regardless of the mode of transmission.
  • CXCR4 — approx. 50% of individuals will have viruses that switch to using this co-receptor. Associated with faster disease progression.
• Activated Cell: Cell must progress past the G1b phase of the cell cycle

Reverse Transcriptase (RT): Encoded by the pol gene, it does not have a proofreading function. As a result, it si very error prone. There is approximately one error “mutation” per round of replication.

Implications of RT error: Approximately one error is made per replication cycle. With up to 1×10⁹ viral particles made per day, it is speculated that every possible mutation is present. As a result, there is tremendous potential for generation of drug resistance. Mutations also allow HIV to escape from immune recognition. Mutations can confer a fitness advantage, be deleterious or make no difference at all to the viral fitness. Areas critical for viral function are conserved, although there is tolerance for variability in areas not as critical and especially those recognized by the immune system.

There is innate and adaptive immunity. Adaptive immunity involves two major types of cells:

So Why Don’t CD8+ T-cells Completely Control HIV?

• Loss of CD4+ T-cells compromises the ability of CTL to work.
• HIV mutation abolishes recognition of HIV by CTL.

How do CTL recognize HIV or other antigens?

• The T-cell receptor on CTL bind short antigen-derived peptides (9-10 amino acids).
• These peptides are presented by proteins on the surface of the cells called Human Leukocyte Antigens (HLA). These are the same proteins that make up your “tissue type”.

Human Leukocyte Antigens:

• Are encoded by the major histocompatibility complex (MHC) on chromosome 6 in humans.
• Class I antigens are found on all nucleated cells. CD8+ T cells recognize Class I antigens.
• Class II antigens are 1° on antigen Presenting cells (macrophages, dendritic cells and B cells). = DR, DP, DQ
• CD4+ T cells recognize Class II antigens.

Viral set-point is indicative of the rate of disease progression. The higher the viral load, the faster the disease progression. It peaks, lowers, plateaus, then increases before terminating.

Long-Term NonProgressors (LTNP)

• ~ 1% of HIV infected individuals
• Infected >10 years, CD4+ T-cells > 500/μl of blood, < 5 X 10³ copies of viral RNA/ml of blood, no clinical disease despite remaining untreated.
• Viral factors: attenuated virus e.g. Nef deletions
• Associated with HLA-B*57, HLA-B*27

HLA Association with LTNP

• Depending on the cohort, HLA-B*57 individuals make up to 85% of LTNP.
• The association with Class I suggests that CTL responses are involved.
• However, HLA can’t be the only factor.
• HLA-B*57 individuals are present among cohorts of “progressors” in the same frequency as the general population.
• HLA-B*57 homozygosity does not necessarily confer LTNP status.
• So why is HLA-B*57 so strongly associated with LTNP status?

HLA-B*57 LTNP Target Conserved Regions Of HIV-1 Derived Proteins

Does this confer better protection from disease progression?
Migueles et al.* have shown that HLA-B*57 progressors also recognize conserved regions of HIV-1 derived proteins. These experiments were all done years after the initial infection.

Is Timing Everything?
The viral set-point is an indicator of the rate of disease progression. The viral set-point is reached early after infection. Shouldn’t we be looking at immune responses early in infection? If LTNP are defined as not progressing to disease progression >10 years, how do you know who to study?

Multi-Center AIDS Cohort Study (MACS)

A cohort of HIV-infected men and at-risk men. Take epidemiological data and biological samples every six months for 20 years. Have individuals who seroconverted while under study. Have HLA-typed many of the men.

Questions We Are Addressing:

• Do CTL from LTNP target conserved regions within HIV earlier than Progressors?
• Early in infection, do CTL from LTNP target more conserved regions than Progressors?
• Is it multi-factorial? For example: maybe CTL all from HLA-B*57 target a high number of conserved regions, but LTNP have other responses that tip the balance in favor of the immune response? Antibodies? Innate immunity?

Results:

• There was no significant difference in the viral load between LTNP and Progressors at the first time-point post-infection. Oddly enough, while both CTL from LTNP and Progressors targeted conserved epitopes, overall, LTNP targeted more non-conserved epitopes than Progressors. Does this help?? During early HIV-1 infection 3/7 LTNP had CTL responses to Env compared to 0/7 progressors.
• 6/7 LTNP vs. 1/7 Progressors targeted an epitope in p24 (capsid).
• The 1 LTNP that did not target this epitope at the earliest time-point, targeted it soon afterwards. He retains that response today, 20 years post-infection. He also still responds to the same epitope in Env as he did 20 years ago.

Caveats:

• To measure responses, we are using peptides that represent the consensus sequences for HIV-1 clade B.
• Even though we have measured CD8+ T-cell responses early in infection, 3 to 9 months post-infection is a long time! Within weeks many other viral infections, like influenza, are won or lost.