February 5: Microbial Genetics: mutation, recombination, complementation.

LECTURE 13: HIV AND AIDS. OVERVIEW OF ANIMAL VIRUSES.

B. HIV = Human Immunodeficiency Virus

1. Receptor attachment

a. Normal function of CD-4:
The CD-4 receptor that HIV recognizes is found on T-Helper cells or (T_H cells). These are cells of the immune system that communicate with antibody-producing B cells. Specifically, T-Helper cells induce the proliferation of the appropriate B cells in response to a specific antigen found on a foreign intruder.

b. Function of CD-4 during infection:
CD-4 interacts with the gp120 protein of HIV, initiating insertion of the viral particle into the T-Helper cells. Infected cells then produce gp120 on their surface and trap other T-Helper cells in large aggregates which fuse to form multi-nucleated syncytia. The syncytia are not viable. Thus the population of T-Helper cells is rapidly reduced.

Recently, a receptor called CCR5 was found to be required for infection of T-Helper cells (in addition to CD-4). Some individuals who have been exposed to HIV do not develop AIDS (Acquired Immune Deficiency Syndrome) because they have a CCR5 variant not recognized by HIV. The virus never penetrates their T-Helper cells, and thus these rare individuals are naturally immune to HIV infection.

c. Diagnostics for AIDS

(i) An individual is considered to have AIDS if his/her CD-4 T-cell count is:

(a) less than 200 per mL blood or

(b) less than 10 % of the total T-cell count

(ii) Opportunistic infections and rare cancers are a consequence of an impaired immune system:

(a) Pneumocystis carinii (a protozoan) infections are common

(b) Kaposi's sarcoma, a rare form of cancer, is common

(c) In women, vaginal infections by Candida albicans (a fungus) are common. In addition, invasive cervical cancers occur.

2. Reverse transcription overview:

All steps are performed by reverse transcriptase, a viral DNA polymerase that can use RNA or DNA as a template.

a. The ssRNA carried in the genome is replicated producing a RNA/DNA hybrid.

b. The RNA portion of this hybrid is degraded by the ribonuclease H activity of reverse transcriptase.

c. The resultant ssDNA is replicated producing dsDNA.

d. Requirements/limitations:

(i) Like all DNA polymerases, reverse transcriptase requires a primer for initiation.

(ii) As with all DNA polymerases, polymerization occurs strictly in the 5'--->3' direction.

e. Replication strategy (see Figure 6.50 for details):

Since the genetic material of retroviruses is linear, a mechanism is needed for preservation of the ends of the molecule. Replication is initially primed using a host t-RNA molecule and later nascent ssDNA "jumps" to a new location so that it can be used as a primer for further DNA synthesis.

3. Integration

The dsDNA produced by reverse transcriptase integrates into the host DNA at random locations. The integrated retroviral genome is called a provirus. The provirus may have a long latency period, i.e. lay dormant for many years, before viral genomes are replicated and packaged into viral particles.

4. Transcription

Retroviral genomes, composed of (+)strand ssRNA, are produced by transcription of the proviral DNA. Host RNA polymerase II and viral-encoded transcription factors are required for transcription. Transcripts are modified by addition of a 5' cap and a 3' poly-A tail using host modification enzymes. These transcripts may be packaged into viral particles or further processed by splicing into mature mRNAs for translation.

5. Translation

Many animal viruses, including retroviruses, produce large precursor "polyproteins" that must be cut at one or more locations in order to generate the final products. Translation products of retroviruses include:

a. gag - a polyprotein whose components include proteins that make up the nucleocapsid coat.

b. gag-pol - an even larger polyprotein resulting from the occasional "erroneous" insertion of an amino acid at the stop codon for gag. The pol portion of this precursor contains the reverse transcriptase protein. This mechanism supplies only a small amount of the enzyme, which is logical since it can act catalytically.

c. Proteases - these are enzymes that cleave the polyprotein precursors.

"Behind every difficulty, there is opportunity." - Albert Einstein

Retroviruses may prove useful in gene therapy. Some retroviruses carry oncogenes. These are genes derived from the host that when mutated or improperly expressed cause cancer. The host copies of these genes have normal functions, often involving the regulation of cell proliferation, e.g. src - a kinase. Thus retroviruses clearly can carry host DNA. They may provide a way to deliver genes into the nucleus of cells since this is a normal step of retroviral "life."

6. Virion production

a. Encapsidation

(i) Viral particles are assembled, but this is not sufficient to produce virions.

(ii) Encapsidated particles interact with the cytoplasmic membrane.

b. Budding
An envelope consisting of a lipid bilayer from the host cytoplasmic membrane is acquired as viral particles are released from the host cell. This is reminiscent of M13. Release of virions from a cell does not necessarily kill the cell.

7. Transmission of HIV

a. Sources: HIV is found in a variety of bodily fluids. Outside of these media, the virus is quite "feeble." This explains why transfer of HIV requires direct contact of such fluids between individuals.

(i) Semen

(ii) Vaginal fluids

(iii) Blood

(iv) Mother's milk

b. High-risk groups:

(i) People who engage in promiscuous unprotected sex, especially anal sex.

(ii) Intravenous drug users who share needles.

(iii) Infants of infected adults.

8. Possible vaccines

a. Soluble CD-4. The strategy is to clone a truncated CD-4 gene into a harmless viral vector that will allow the extracellular portion of CD-4 to be expressed in the bloodstream. The hope is that the gp120 glycoprotein on HIV will interact with these "soluble" CD-4 fragments and leave the native CD-4 receptor on the T-Helper cells alone, thus averting infection.

b. Soluble CCR5. The idea is the same as the soluble CD-4 strategy.

c. Anti-idiotypic antibodies to gp120. Antibodies directed against gp120 should resemble CD-4 if they recognize the same structural features of gp120 that CD-4 does. An antibody subsequently raised against the idiotype of the anti-gp120 antibody will resemble gp120 for similar reasons. This product could be used to immunize people and thus prime them to destroy intruders bearing the real gp120.

9. Currently available treatments

a. Chain terminators. Administration of nucleotide analogues that prematurely terminate nucleic acid replication will prevent the synthesis of viral genomes and may thus slow down the production of HIV virions. Chain terminators include dideoxynucleotidetriphosphates (ddNTPs - the same molecules used in Sanger's DNA sequencing procedure) and AZT which has a 3' azido group instead of a hyroxyl group. A concern - host DNA synthesis is poisoned as well as viral replication.

b. Protease inhibitors. The polyproteins of HIV must be cleaved by proteases if new viral particles are to be produced. Thus the administration of small molecules that inhibit protease activity should slow down virion production.

Concerns - host proteases must not be poisoned.

Currently, the best approach is to hit HIV on several fronts by using several chain terminators in combination with a protease inhibitor. The virus, however, is notorious for its ability to develop resistance to treatments. Therefore patients must be carefully monitored to determine the efficacy of the therapy. If resistance occurs, the types of drugs being administered must be switched immediately.

10. Some treatments under development

a. Antisense oligonucleotides. The strategy is to administer oligonucleotides complementary to HIV sequences. By hybridizing with their target, these molecules are expected to interfere with replication of the viral genome or with the translation of key genes on the (+)strand RNA. Efficient delivery of these oligonucleotides to the infected cells is still a stumbling block.

b. Blocking of RNA structure. Gabriele Varani (1996) - has developed methods to determine the tertiary structure of RNA by nuclear magnetic resonance (NMR). He has examined the structure of HIV RNA in hope of finding a point to attack the virus. Specifically, it is known that interaction of the TAR sequence on the "second" HIV genome with the viral protein Tat activates transcription of viral genes by RNA polymerase II. Varani has solved the structure of the "free" TAR RNA region and the Tat-bound TAR RNA region. He is using this structural information to design small organic molecules that will lock the TAR RNA region in the unbound conformation and thus prevent the activation of viral gene expression.

I. Overview of animal viruses

A. Some features already encountered in bacteriophages:

Transposition - for integration of the viral genome into the host DNA, e.g. Mu; HIV.
Budding - release of virions without killing the host cell, e.g. M13; HIV, rabies virus, influenza virus.
Replicases - RNA-dependent RNA polymerases for synthesizing complementary RNA, e.g. MS2; poliovirus, rabies virus.
Overlapping genes e.g. PhiX174, MS2; SV40.
Rolling circle replication, e.g. PhiX174, lambda; herpes virus.

B. Some new lessons/surprises from animal viruses:

Reverse transcription, e.g. HIV and other retroviruses.
Double-stranded RNA, e.g. rotavirus.
Protein primers for initiating DNA replication, e.g. adenovirus.
Scavenging of host macromolecules for the virion, e.g. HIV scavenges host t-RNAs, SV40 scavenges host histones.
Multiple roles for RNA, e.g. HIV uses RNA as its genetic material and as a transcriptional co-activator, e.g. self-splicing catalytic RNAs.
Segmented genomes, e.g. the genome of rotavirus consists of 10-12 segments.
Production of polyproteins e.g. HIV, poliovirus.

C. Two major challenges posed to viruses by the eukaryotic translation machinery:

The mRNAs of eukaryotes are recognized based on structural modifications that occur in the nucleus. Unlike prokaryotes, eukaryotes do not use ribosome binding sites to inform the translation apparatus of the location of sequences that need to be translated. Instead, initiation relies on the recognition of the 5' Cap by the CAP-binding complex. Thus, if viral messages are to be translated by the eukaryotic machinery, they must find a way to be recognized as such.
The mRNAs of eukaryotes are monocistronic. A cistron is the information in mRNA encoding one protein. In prokaryotes, a single mRNA may be polycistronic, i.e. it may contain several different open reading frames, each having its own ribosome binding site. Thus, a bacteriophage could encode multiple proteins on a single mRNA. In eukaryotes, each mature mRNA generally contains only one open reading frame, encoding only one protein. Eukarytic viruses must find some way around this if they are to express multiple proteins within their host cells using a single transcript.

D. Viral solutions to the first major challenge - modified mRNAs.

1. If the virus is strictly cytoplasmic (i.e. it never enters the nucleus):

2. If the virus enters the nucleus:

a. The virus may simply bring along its own modification enymes (a capping enzyme and a poly-A polymerase) and thus prepare its mRNA(s) for cytoplasmic translation. E.g. rotavirus, vaccinia virus.

b. The virus may destroy the host Cap-binding protein complex, loosening specificity of the protein synthesis initiation process and allowing the translation of proteins encoded on modified viral mRNAs. e.g. poliovirus modifies the 5' ends of its mRNAs by attaching a peptide, allowing them to be translated without being capped.

a. The virus may simply exploit the host modification machinery by allowing the host to modify its mRNAs after they are produced in the nucleus. E.g. HIV, SV40.

b. The virus may scavenge 5' Caps from host mRNAs and transfer them to viral mRNAs. The process requires an RNA endonuclease. This simultaneously disables host messages and allows viral messages to be translated by the eukaryotic translation apparatus. E.g. Influenza virus.

D. Viral solutions to the second major challenge - monocistronic mRNAs.

1. If the virus is strictly cytoplasmic:

2. If the virus enters the nucleus:

a. Segmented genomes allow some viruses to produce several different monocistronic mRNAs, thus allowing the production of multiple proteins.

b. Some viruses use multiple translation start sites in a single mRNA, thus allowing more than one protein to be produced from that mRNA. In eukaryotic translation, only one start site normally occurs, located at the first AUG after the 5' cap. However, some eukaryotic viruses induce the translation machinery to use other AUG start codons in addition to the 5' proximal one.

c. Similarly, some viruses use alternative translation stop sites by inducing the translation apparatus to "read-through" or jump over the first stop codon encountered a certain percentage of the time.

d. The production of polyproteins that are processed by proteases yielding several component proteins allows some viruses to make multiple proteins from one monocistronic mRNA.

a, b, c, d. The same strategies used by cytoplasmic viruses may also be used. E.g. HIV uses a "read-through" mechanism (D.1.c) to produce the gag-pol polyprotein (D.1.d).

e. Alternative splicing - different combinations of exons may be spliced together when RNA transcripts are spliced. This allows the virus to encode more than one protein from a single primary mRNA transcript.