HIV: Structure, Laboratory Diagnosis, and Natural Resistance

Human immunodeficiency virus (HIV) is a complex RNA retrovirus that causes acquired immunodeficiency syndrome (AIDS) — one of the most significant infectious diseases in human history. Understanding HIV from a microbiological perspective requires three things: knowing how the virus is built, knowing how infection is diagnosed in the laboratory, and understanding why some individuals are naturally protected from infection.

Part 1 — Structure of HIV

HIV belongs to the genus Lentivirus within the family Retroviridae. Two types exist — HIV-1 (the predominant global cause of AIDS) and HIV-2 (less virulent, mainly found in West Africa). The virus is approximately 100–120 nm in diameter with a characteristic dense, cone-shaped nucleocapsid.

Overall structure

HIV has the following structural layers from outside to inside:

1. Lipid bilayer envelope The outermost layer is derived from the host cell membrane during the budding process. It contains both viral glycoproteins and host cell proteins acquired during budding.

2. Envelope glycoproteins (Env) — gp120 and gp41 72 external spikes are embedded in the envelope, each composed of two major glycoproteins:

gp120 (surface unit) — binds to the CD4 receptor on CD4+ T lymphocytes and cells of the monocyte/macrophage lineage. Also binds to chemokine coreceptors CCR5 and CXCR4. HIV-1 and HIV-2 are distinguished serologically by differences in gp120. On the basis of gp120 gene variability (V3 region), HIV-1 is further classified into subtypes (clades A through I)
gp41 (transmembrane unit) — mediates fusion between the viral membrane and the host cell membrane, allowing viral entry

3. Matrix protein (p17) Beneath the viral envelope, p17 maintains the structural integrity of the virion and plays a role in nuclear transport of the pre-integration complex.

4. Capsid (p24) A protein shell enclosing the viral RNA and associated enzymes. The p24 antigen is detectable in blood during acute infection before antibodies develop — the basis of the p24 antigen test in early HIV diagnosis.

5. Nucleocapsid proteins (p7, p9) Enclose the viral RNA genome within the capsid during assembly.

6. Viral RNA genome Two identical copies of a 9.8 kb single-stranded positive-polarity RNA genome. HIV is a retrovirus — its RNA genome must be reverse transcribed into DNA before integration into the host genome.

Viral enzymes

Three enzymes are packaged inside the HIV virion, all encoded by the pol gene and all targets of antiretroviral therapy:

Enzyme	Function	Drug class targeting it
Reverse transcriptase (RT)	Converts viral RNA genome into double-stranded DNA	NRTIs, NNRTIs
Integrase (IN)	Integrates viral DNA into host cell chromosome	Integrase inhibitors (raltegravir, dolutegravir)
Protease (PR)	Cleaves viral polyproteins into functional proteins during virion maturation	Protease inhibitors (ritonavir, lopinavir)

Structural and regulatory genes

HIV encodes 3 structural genes and 6 regulatory genes:

Structural genes:

Gag — encodes core proteins: p24 (capsid), p17 (matrix), p7/p9 (nucleocapsid)
Env — encodes envelope glycoproteins: gp120 and gp41
Pol — encodes viral enzymes: reverse transcriptase, integrase, protease

Regulatory genes (required for replication):

Tat — activates transcription of viral genes
Rev — transports late mRNAs from nucleus to cytoplasm

Regulatory genes (not required for replication):

Nef — decreases CD4 and MHC class I expression on infected cells. Deletions/mutations in the nef gene are found in some long-term non-progressors — individuals infected with HIV who do not progress to AIDS
Vif — enhances viral infectivity
Vpr — transports viral core from cytoplasm into nucleus
Vpu — enhances virion release from the cell

Part 2 — Laboratory Diagnosis of HIV Infection

Laboratory tests for HIV diagnosis are divided into four categories: antigen/antibody detection, virus cultivation, antibody confirmation, and viral genome amplification.

Sample collection and transport

Standard HIV-1/HIV-2 antibody testing requires a single tube (10 mL) of whole blood
Specimens stored at room temperature: stable for up to 3 days
Specimens stored at 4°C: stable for up to 7 days
For longer storage, serum or plasma must be separated and stored at −20°C
Specimens for PCR must be processed within 48 hours — viral RNA/DNA degrades over time

Window period

Antibodies to HIV become detectable 3 to 12 weeks after infection. This gap between infection and antibody detectability is the window period — a critical time when a recently infected person may test antibody-negative despite carrying the virus. Modern 4th-generation combination antigen/antibody tests have reduced the window period to less than 3 weeks by detecting both p24 antigen (present from day 14–21) and HIV antibodies simultaneously.

Diagnostic algorithm

Step 1 — Screening test: 4th generation ELISA (combination Ag/Ab assay) The current standard for HIV screening. Detects both HIV-1/HIV-2 antibodies and HIV-1 p24 antigen simultaneously. Highly sensitive (>99.5%) but false positives occur. Any reactive result must be confirmed.

Step 2 — Confirmatory test: Western blot or HIV-1/HIV-2 differentiation immunoassay A positive Western blot requires bands at specific HIV protein positions — at minimum, two bands from: p24, gp41, gp120/160. An indeterminate Western blot requires further follow-up testing. Modern HIV differentiation immunoassays (identifying HIV-1 vs HIV-2) are now preferred over Western blot in many reference laboratories.

Step 3 — Nucleic acid test (NAT/PCR) for indeterminate or discordant results

Individual diagnostic tests in detail

Point of care (rapid) tests Immunochromatographic tests that can be performed in 15–30 minutes at the bedside or in resource-limited settings. Detect HIV-1/HIV-2 antibodies. High sensitivity and specificity but all positives must be confirmed with laboratory testing. Widely used in antenatal screening programs and voluntary counseling and testing centers.

ELISA (Enzyme-Linked Immunosorbent Assay) The most widely used HIV screening test. 4th generation assays detect both p24 antigen and IgM/IgG antibodies. False positives can occur in autoimmune conditions, pregnancy, malaria, and recent influenza vaccination. All repeatedly reactive ELISA results must be confirmed by Western blot or NAT.

p24 Antigen testing p24 antigen appears in blood 14–21 days after infection — before antibodies develop. Particularly useful:

For patients who are high risk and symptomatic but HIV antibody-negative (early acute infection during window period)
For specimens that are ELISA-positive but Western blot-negative or indeterminate
For confirming HIV infection in neonates born to HIV-positive mothers — maternal IgG antibodies cross the placenta and make antibody-based testing unreliable in infants under 18 months

Western blot Highly specific confirmatory test. Viral proteins are separated by electrophoresis, transferred to a membrane, and probed with patient serum. Specific antibody bands at positions corresponding to HIV structural proteins (gp160, gp120, gp41, p66, p51, p31, p24, p17) indicate HIV infection. A positive result requires reactivity at a minimum of two envelope bands.

HIV PCR (Nucleic Acid Amplification Test) Detects HIV RNA (viral load test) or HIV proviral DNA in blood. Uses reverse transcription PCR (RT-PCR). Key applications:

Diagnosis in infants — PCR is the standard for diagnosing HIV in infants born to HIV-positive mothers, tested at 14–21 days, 1–2 months, and 4–6 months of age
Resolving indeterminate Western blot results
Testing immunocompromised patients who may not mount an antibody response
Viral load monitoring — quantitative HIV RNA PCR monitors disease progression and treatment response in HIV-positive patients

Virus cultivation HIV can be isolated from peripheral blood mononuclear cells (PBMCs) co-cultivated with activated PBMCs from HIV-negative donors in the presence of IL-2. A positive result is detected by p24 antigen or reverse transcriptase activity in the culture medium. This is not performed in routine diagnostic laboratories — used only in research settings.

CD4+ lymphocyte count

A hallmark of chronic HIV infection is the progressive depletion of CD4+ T lymphocytes. CD4 count monitoring is essential for:

Clinical staging of HIV infection
Deciding when to initiate antiretroviral therapy
Initiating prophylaxis against opportunistic infections (e.g. Pneumocystis jirovecii pneumonia prophylaxis when CD4 < 200 cells/μL)
Monitoring treatment response

CD4 count	Clinical significance
> 500 cells/μL	Normal or early infection; no opportunistic infections
200–500 cells/μL	Moderate immunosuppression; increased risk
< 200 cells/μL	Severe immunosuppression — AIDS-defining threshold; PCP prophylaxis indicated
< 50 cells/μL	Profound immunosuppression; high risk of CMV, MAC, CNS lymphoma

Part 3 — Natural Resistance to HIV Infection

Not everyone exposed to HIV becomes infected. Some individuals — particularly those repeatedly exposed through high-risk behavior — remain HIV-negative. While safe sex practices and needle safety explain many cases, a subset of people appear to have genetic protection against HIV infection.

How HIV enters a cell — the receptor problem

For a virus to infect a cell, it must first attach firmly to it. HIV requires two sequential binding steps to enter a CD4+ T lymphocyte:

Primary receptor binding: gp120 on the HIV surface binds to the CD4 receptor on the host cell. This causes a conformational change in gp120 that exposes the coreceptor binding site
Coreceptor binding: The exposed gp120 then binds to a chemokine coreceptor — either CCR5 or CXCR4 — on the same cell surface
Membrane fusion: gp41 mediates fusion between viral and cellular membranes, allowing viral entry

If either the CD4 receptor or the required coreceptor is absent from the cell surface, HIV cannot enter — the entire infection process stops at step one.

HIV uses different coreceptors at different stages:

Early infection: HIV is predominantly R5-tropic — uses CCR5 coreceptor. Transmitted via sexual contact, blood, or vertical transmission
Late infection/AIDS: HIV may switch to X4-tropic (CXCR4) or dual-tropic (both), correlating with more rapid CD4 decline

The CCR5-Δ32 mutation — genetic resistance to HIV

Studies have found that a specific mutation in the gene encoding CCR5 — called CCR5-Δ32 (delta 32, a 32-base pair deletion) — produces a truncated, non-functional CCR5 protein that is not expressed on the cell surface. Without surface CCR5, R5-tropic HIV (which accounts for almost all sexually transmitted HIV) cannot enter CD4+ T cells.

The clinical significance:

Homozygotes (two copies of CCR5-Δ32): Essentially completely resistant to R5-tropic HIV infection. Even with repeated high-risk exposure, these individuals do not become infected with the predominant strains of HIV
Heterozygotes (one copy of CCR5-Δ32): Not fully protected from infection, but if infected, disease progresses significantly more slowly — lower viral loads, slower CD4 decline, longer time to AIDS

This mutation is found predominantly in people of Western European ancestry:

~1% are homozygous (fully resistant)
~10–15% are heterozygous (partial protection)

The mutation is rare in East Asian, African, and South Asian populations, which may partly explain regional differences in HIV transmission dynamics.

Clinical application — CCR5 inhibitors

The discovery of CCR5 as an essential HIV coreceptor led directly to the development of CCR5 inhibitor antiretroviral drugs — a class of drugs that block the CCR5 receptor on CD4+ cells, preventing R5-tropic HIV from attaching. Maraviroc is the first approved CCR5 inhibitor (2007), used in patients with confirmed R5-tropic HIV infection.

### The Berlin Patient — first cure of HIV

Timothy Ray Brown, known as "The Berlin Patient," is recognized as the first person functionally cured of HIV.

Brown was diagnosed with HIV in 1995. In 2006, he was also diagnosed with acute myeloid leukemia. His German physicians made a remarkable treatment decision — they selected a bone marrow donor who was homozygous for CCR5-Δ32.

After two stem cell transplants from this CCR5-Δ32 homozygous donor:

His leukemia was cured
His immune system was reconstituted with donor cells lacking functional CCR5
HIV was undetectable in his blood and tissues — and remained so until his death from leukemia relapse in 2020

Brown's case proved that eliminating CCR5 expression from immune cells can effectively cure HIV infection, and it remains the foundational case study for ongoing gene therapy and stem cell approaches to HIV cure research.

Brown later founded the Timothy Ray Brown Foundation in Washington, DC, dedicated to HIV/AIDS cure research.

References and Further Reading

Tille, P. M. (2017). Bailey & Scott's Diagnostic Microbiology (14th ed.). Mosby Elsevier.
Turner, B. G., & Summers, M. F. (1999). Structural biology of HIV. Journal of Molecular Biology, 285(1), 1–32. https://doi.org/10.1006/jmbi.1998.2354
Buttò, S., Suligoi, B., Fanales-Belasio, E., & Raimondo, M. (2010). Laboratory diagnostics for HIV infection. Annali dell'Istituto superiore di sanità, 46(1), 24–33. https://doi.org/10.4415/ANN_10_01_04
Fearon, M. (2005). The laboratory diagnosis of HIV infections. Canadian Journal of Infectious Diseases and Medical Microbiology, 16(1), 26–30. https://doi.org/10.1155/2005/515063
Samson, M., Libert, F., Doranz, B. J., et al. (1996). Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature, 382(6593), 722–725. https://doi.org/10.1038/382722a0
Biasin, M., De Luca, M., Gnudi, F., & Clerici, M. (2013). The genetic basis of resistance to HIV infection and disease progression. Expert Review of Clinical Immunology, 9(4), 319–334. https://doi.org/10.1586/eci.13.16
WHO. (2019). Consolidated Guidelines on HIV Testing Services. World Health Organization.