HIV

Question 1

HIV Structure

Answer 1

A. The viral envelope is formed from the host cell
membrane, and contains HIV-1 envelope proteins gp41 and gp120 and several host cell proteins, e.g., major histocompatibility complex (MHC) class II proteins.
B. The matrix between the envelope and the core is mainly Gag protein p17.
C. The core contains the viral RNA, Gag protein p7, reverse transcriptase (RT) and integrase (IN). Virions contain complementary DNA, synthesized by the RT. The major structural proteins of the core are Gag proteins p24 and p6.
D. Vpr and two cleavage products from the Gag precursor protein (p1 and p2, not shown) are also found within the virion.

Question 2

Describe the morphology of HIV and how its classified

Answer 2

• Morphologic distinction of retrovirions.
• The morphology and position of the nucleocapsid
  core are used to classify the viruses.
• A-type particles are immature intracytoplasmic
  forms that bud through the plasma membrane and
  mature into B-type, C-type, and D-type particles.

Question 3

How does HIV infect cells

Answer 3

A. Virions breach the epithelial barrier during sex, and in the submucosa use their gp120 to bind to CD4 and co-receptors CXCR4 or CCR5 on Th cells, DCs and Mϕ

B. Fusion with host cell membrane releases viral core into cytoplasm where it is transported to the nucleus.

Question 4

How does HIV replicate?

Answer 4

Cytoplasm and nucleus are both involved to create lifelong infection.

A. Once released into the cytoplasm, each ss(+)RNA is read by viral reverse transcriptase (RT) to make a copy of complimentary DNA (cDNA). The ssRNA is destroyed in the process. The RT then makes a complimentary copy of the cDNA to produce a double-stranded cDNA molecule.
B. The double-stranded cDNA is then transported into the nucleus of the cell and an integrase enzyme (IN), encoded by HIV1, clips and inserts the cDNA into the host chromosome, creating a provirus. Another enzyme, Vpr, can do this too, especially in non-proliferative cells like Mϕ.
C. Integrated proviral DNA is
transcribed by host RNA
polymerase (RNAP) to make new viral RNA genomes, and into mRNA to translate new viral proteins (not shown!).

**Note that HIV replication only occurs in actively dividing (proliferating)
cells. Thus the proviral genome is mainly silent in Th cells and Mϕ that are normally resting. This provides a long-term source of latent virus.

Question 5

What are 3 defensive strategies of HIV?

Answer 5

HIV uses both defensive and offensive strategies to evade the immune system

Three defensive strategies:
1. It only takes HIV-1 five to ten days to establish latent infection, i.e., before the
adaptive immunity can respond. Latency does not attract an immune response
and insures virus infection for the life of its host.

2. Viruses can be passed directly from cell to cell by “transfection”.
3. High mutation rate of reverse transcriptase creates progeny viruses that are
   different than the viruses that infected the cell. This keeps the virus antigenically
   ahead of the adaptive immune system’s CTLs or antibodies when the adaptive
   immune response is activated once HIV proviruses begin to replicate.

Question 6

What are HIVs offensive strategies against the immune system?

Answer 6

A: There are two main lineages of DCs in humans, the myeloid DCs (MDCs, also named classical or conventional DCs) and plasmacytoid DCs (PDCs). MDCs are originated from a myeloid progenitor in the bone marrow, while PDCs may come from a lymphoid progenitor in lymphoid organs.Infection of PDCs by HIV-1 leads to interferon
production and subsequently, the interferonstimulated gene (ISG) product, APOBEC3G.
This protein is designed to cause potentially lethal mutations in the proviral DNA. But HIV1 protein Vif, destroys APOBEC3G.

B: Problem: HIV-1 mainly infects immune cells that do not proliferate (divide). If a cell doesn’t divide, the proviral DNA isn’t translated or transcribed, and no new virus is produced. The problem of viral proliferation is solved by turning immunity against itself. Infection of PDCs by HIV-1 leads to interferon production that also stimulates Th cells to
divide. This division of Th cells infected with provirus leads to propagation of new virus. Note: any cell with a latent infection will also tend to make new virus when it divides.
When HIV-1 infects conventional DCs, (MDCs) they get transported to the lymph nodes where there are lots of proliferating T cells and Mϕ for HIV-1 to infect and use as “virus factories”.
Afterward, HIV-1 either kills the Th cells it infects or marks them for destruction by CTLs, depleting their numbers and crippling the adaptive immune system for lack of T help.

Question 7

Describe the pathogenic events in untreated HIV-mediated disease.

Answer 7

1. HIV (pink spheres, red arrows) enters the body and binds to Langerhans or dendritic cells (orange), which carry the virus to CD4+ T cells.
2. Infected CD4+ T cells home to lymphoid tissue, where the infection is established.
3. Virus replication accelerates, and massive viremia leads to the wide dissemination of virus throughout the body’s lymphoid tissue.
4. An HIV-specific immune response occurs and virus is trapped on the follicular dendritic cells of germinal centers in the lymphoid
   tissue.
5. At this point, chronic, persistent infection is established despite an
   immunological response to the virus.
6. Immune activation is an important driver of HIV replication and is mediated by the secretion of various cytokines and by aberrant cell signaling caused by interaction of the viral envelope with cellular
   receptors.
7. Because there is usually only partial immunological control of virus replication, continual and accelerated production of virus ensues.
   (This is associated with a rapid turnover of CD4+ T cells.)
8. Ultimately, lymphocyte depletion occurs, along with destruction of the architecture of lymphoid tissue

Question 8

How does HIV spread?

Answer 8

• Infectivity of HIV-1 through
  intercourse is not very high if it’s between an otherwise healthy individual and an uninfected partner. (1 in 1000 or less).
• Other STIs (e.g., H. ducreyi,
  syphilis, herpes, etc.) contribute to infectivity by creating open sores and recruiting CD4+ cells through inflammation.
• Rectal epithelium is vulnerable to tearing during anal intercourse, providing access to HIV-1.
• Larger number of partners
  contributes to spread.
• Intravenous drug users who
  share needles (e.g., ~50% of
  HIV+ women in the U.S. today
  acquired their virus this way)

Question 9

Is there a cure for HIV?

Answer 9

No

Question 10

Describe how you can diagnose HIV?

Answer 10

• First line screening – uses an ELISAbased assay to detect antibodies against HIV-1 and HIV-2 and the HIV-1 early antigen (p24)
• Antibody Differentiation assay – confirmation assay which detects HIV-1 and HIV-2 antibodies
• Viral load testing (or nucleic acid testing (NAT)) – generally most labs have HIV-1 NATs, although some have combined 1 and 2 NATs
• Resistance testing – sequences the HIV genome and gives a genotype, presence of resistance markers and looks at the integrase inhibitor resistance

Question 11

HIV reflex testing

Answer 11

• Reflex testing – uses a
  single sample to perform
  the ELISA-based assay and the NAT assay for confirmation
• A second sample is now
  no longer required to confirm a result
• We are now working on
  including resistance
  testing off the same tube
• This would allow improved linkage to care and would give all testing needed to start therapy from the first sample

Question 12

What do we hope to accmplish with reflex testing?

Answer 12

• ~97% of individuals
  received a definitive diagnosis with a single
  blood sample
• Turnaround times
  decreased from 39 days to 4 days

Question 13

What do we hope will happen for HIV cascade of care?

Answer 13

• A reduced turnaround time from diagnosis to molecular
  confirmation
• TATs have decreased from 41 to 4 days
• A reduction between time of
  diagnosis and specialty care and treatment
• Improved retention in care