RESUMEN
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) dual infection (DI) has been associated with decreased CD4 T-cell counts and increased viral loads; however, the frequency of intrasubtype DI is poorly understood. We used ultradeep sequencing (UDS) to estimate the frequency of DI in a primary infection cohort of predominantly men who have sex with men (MSM). METHODS: HIV-1 genomes from longitudinal blood samples of recently infected, therapy-naive participants were interrogated with UDS. DI was confirmed when maximum sequence divergence was excessive and supported by phylogenetic analysis. Coinfection was defined as DI at baseline; superinfection was monoinfection at baseline and DI at a later time point. RESULTS: Of 118 participants, 7 were coinfected and 10 acquired superinfection. Superinfection incidence rate was 4.96 per 100 person-years (95% confidence interval [CI], 2.67-9.22); 6 occurred in the first year and 4 in the second. Overall cumulative prevalence of intrasubtype B DI was 14.4% (95% CI, 8.6%-22.1%). Primary HIV-1 incidence was 4.37 per 100 person-years (95% CI, 3.56-5.36). CONCLUSIONS: Intrasubtype DI was frequent and comparable to primary infection rates among MSM in San Diego; however, superinfection rates declined over time. DI is likely an important component of the HIV epidemic dynamics, and development of stronger immune responses to the initial infection may protect from superinfection.
Asunto(s)
Coinfección/epidemiología , Coinfección/virología , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , Adulto , Femenino , Genotipo , VIH-1/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Homosexualidad Masculina , Humanos , Incidencia , Masculino , Filogenia , Prevalencia , ARN Viral/genética , Estados Unidos/epidemiologíaRESUMEN
Investigating the incidence and prevalence of HIV-1 superinfection is challenging due to the complex dynamics of two infecting strains. The superinfecting strain can replace the initial strain, be transiently expressed, or persist along with the initial strain in distinct or in recombined forms. Various selective pressures influence these alternative scenarios in different HIV-1 coding regions. We hypothesized that the potency of the neutralizing antibody (NAb) response to autologous viruses would modulate viral dynamics in env following superinfection in a limited set of superinfection cases. HIV-1 env pyrosequencing data were generated from blood plasma collected from 7 individuals with evidence of superinfection. Viral variants within each patient were screened for recombination, and viral dynamics were evaluated using nucleotide diversity. NAb responses to autologous viruses were evaluated before and after superinfection. In 4 individuals, the superinfecting strain replaced the original strain. In 2 individuals, both initial and superinfecting strains continued to cocirculate. In the final individual, the surviving lineage was the product of interstrain recombination. NAb responses to autologous viruses that were detected within the first 2 years of HIV-1 infection were weak or absent for 6 of the 7 recently infected individuals at the time of and shortly following superinfection. These 6 individuals had detectable on-going viral replication of distinct superinfecting virus in the env coding region. In the remaining case, there was an early and strong autologous NAb response, which was associated with extensive recombination in env between initial and superinfecting strains. This extensive recombination made superinfection more difficult to identify and may explain why the detection of superinfection has typically been associated with low autologous NAb titers.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Evolución Biológica , Infecciones por VIH/virología , VIH-1/genética , Sobreinfección/virología , Adulto , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/clasificación , VIH-1/aislamiento & purificación , VIH-1/fisiología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Recombinación Genética , Sobreinfección/inmunología , Adulto Joven , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
HIV-1 dual infection (DI) and CXCR4 (X4) coreceptor usage are associated with accelerated disease progression but frequency and dynamics of coreceptor usage during DI is unknown. Ultradeep sequencing was used to interrogate for DI and infer coreceptor usage in longitudinal blood samples of 102 subjects. At baseline, X4 usage was high (23 subjects harbored X4 variants) and was not associated with infection duration or DI. Coreceptor usage changed over time in 12 of 47 participants, and X4 usage emerged in 4 of 41 monoinfections vs 2 of 5 superinfections (P = .12), suggesting a weak statistical trend toward occurrence of superinfection and acquiring X4 usage.
Asunto(s)
Infecciones por VIH/virología , VIH-1/genética , Adulto , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Masculino , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Análisis de Secuencia de ADN/métodosRESUMEN
We sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of â¼3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in ≥0.5% of UDPS reads in a sample. Sanger sequencing identified ≥1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%; P = 0.06). UDPS detected ≥1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%; P = 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P < 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.
Asunto(s)
Antivirales/uso terapéutico , ADN Viral/sangre , Farmacorresistencia Viral/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Hepatitis B Crónica/virología , Lamivudine/uso terapéutico , Mutación , Adulto , Antivirales/farmacología , Esquema de Medicación , Farmacorresistencia Viral/genética , Femenino , Virus de la Hepatitis B/crecimiento & desarrollo , Hepatitis B Crónica/sangre , Hepatitis B Crónica/tratamiento farmacológico , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lamivudine/farmacología , Masculino , Persona de Mediana Edad , Tipificación Molecular , Estudios Retrospectivos , Factores de Tiempo , Carga ViralRESUMEN
OBJECTIVES: To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs. METHODS: We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing. RESULTS: For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture. CONCLUSIONS: The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations.
Asunto(s)
Farmacorresistencia Viral , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Mutación Missense , VIH-1/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Plasma/virología , ARN Viral/genéticaRESUMEN
Reports of a high frequency of the transmission of minority viral populations with drug-resistant mutations (DRM) are inconsistent with evidence that HIV-1 infections usually arise from mono- or oligoclonal transmission. We performed ultradeep sequencing (UDS) of partial HIV-1 gag, pol, and env genes from 32 recently infected individuals. We then evaluated overall and per-site diversity levels, selective pressure, sequence reproducibility, and presence of DRM and accessory mutations (AM). To differentiate biologically meaningful mutations from those caused by methodological errors, we obtained multinomial confidence intervals (CI) for the proportion of DRM at each site and fitted a binomial mixture model to determine background error rates for each sample. We then examined the association between detected minority DRM and the virologic failure of first-line antiretroviral therapy (ART). Similar to other studies, we observed increased detection of DRM at low frequencies (average, 0.56%; 95% CI, 0.43 to 0.69; expected UDS error, 0.21 ± 0.08% mutations/site). For 8 duplicate runs, there was variability in the proportions of minority DRM. There was no indication of increased diversity or selection at DRM sites compared to other sites and no association between minority DRM and AM. There was no correlation between detected minority DRM and clinical failure of first-line ART. It is unlikely that minority viral variants harboring DRM are transmitted and maintained in the recipient host. The majority of low-frequency DRM detected using UDS are likely errors inherent to UDS methodology or a consequence of error-prone HIV-1 replication.
Asunto(s)
Farmacorresistencia Viral/genética , Infecciones por VIH/transmisión , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , Adulto , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Secuencia de Bases , Femenino , Genes env , Genes gag , Genes pol , Variación Genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/crecimiento & desarrollo , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/genética , Análisis de Secuencia de ARN , Resultado del Tratamiento , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaAsunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones Oportunistas/etiología , Infecciones por Retroviridae/complicaciones , Retroviridae , Fármacos Anti-VIH/efectos adversos , Fármacos Anti-VIH/farmacocinética , Terapia Antirretroviral Altamente Activa/efectos adversos , Infecciones por VIH/complicaciones , Humanos , Malaria/complicaciones , Infecciones Oportunistas/virología , Farmacogenética , Infecciones por Retroviridae/virologíaRESUMEN
OBJECTIVE: Characterize intra-individual HIV-1 subtype B pol evolution in antiretroviral naive individuals. DESIGN: Longitudinal cohort study of individuals enrolled during primary infection. METHODS: Eligible individuals were antiretroviral naïve participants enrolled in the cohort from December 1997-December 2005 and having at least two blood samples available with the first one collected within a year of their estimated date of infection. Population-based pol sequences were generated from collected blood samples and analyzed for genetic divergence over time in respect to dual infection status, HLA, CD4 count and viral load. RESULTS: 93 participants were observed for a median of 1.8 years (Mean = 2.2 years, SD =1.9 years). All participants classified as mono-infected had less than 0.7% divergence between any two of their pol sequences using the Tamura-Nei model (TN93), while individuals with dual infection had up to 7.0% divergence. The global substitution rates (substitutions/nucleotide/year) for mono and dually infected individuals were significantly different (p<0.001); however, substitution rates were not associated with HLA haplotype, CD4 or viral load. CONCLUSIONS: Even after a maximum of almost 9 years of follow-up, all mono-infected participants had less than 1% divergence between baseline and longitudinal sequences, while participants with dual infection had 10 times greater divergence. These data support the use of HIV-1 pol sequence data to evaluate transmission events, networks and HIV-1 dual infection.
Asunto(s)
Genes Virales/genética , Genes pol/genética , Infecciones por VIH/genética , VIH-1/genética , Adulto , Terapia Antirretroviral Altamente Activa/métodos , Recuento de Linfocito CD4/métodos , Estudios de Cohortes , Evolución Molecular , Femenino , Variación Genética/genética , Infecciones por VIH/tratamiento farmacológico , Antígenos HLA/genética , Humanos , Estudios Longitudinales , Persona de Mediana Edad , Carga Viral/métodos , Adulto JovenRESUMEN
OBJECTIVE: To investigate the susceptibilities to and consequences of HIV-1 dual infection. DESIGN: We compared clinical, virologic, and immunologic factors between participants who were dually infected with HIV-1 subtype B and monoinfected controls who were matched by ongoing HIV risk factor. METHODS: The viral load and CD4 progressions of dually and singly infected participant groups were compared with linear mixed-effects models, and individual dynamics before and after superinfection were assessed with a structural change test (Chow test). Recombination breakpoint analysis (GARD), HLA frequency analysis, and cytotoxic T-lymphocyte (CTL) epitope mapping were also performed (HIV LANL Database). RESULTS: The viral loads of dually infected participants increased more over 3 years of follow-up than the viral loads of monoinfected controls, whereas CD4 progressions of the two groups did not differ. Viral escape from CTL responses following superinfection was observed in two participants whose superinfecting strain completely replaced the initial strain. This pattern was not seen among participants whose superinfecting virus persisted in a recombinant form with the initial virus or was only detected transiently. Several HLA types were over-represented in dually infected participants as compared to monoinfected controls. CONCLUSIONS: These results identify potential factors for dual infection susceptibility and further define its clinical consequences.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/aislamiento & purificación , Homosexualidad Masculina , Inmunofenotipificación , Sobreinfección/inmunología , Linfocitos T Citotóxicos/inmunología , Adulto , Algoritmos , Recuento de Linfocito CD4 , Estudios de Casos y Controles , Progresión de la Enfermedad , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/inmunología , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , ARN Viral , Sobreinfección/virología , Linfocitos T Citotóxicos/virología , Carga Viral , Adulto JovenRESUMEN
Current methods to detect intraclade HIV dual infection are poorly suited for determining its prevalence in large cohorts. To investigate the potential of ultra-deep sequencing to screen for dual infection, we compared it to bulk sequence-based synonymous mixture index and the current standard of single genome sequencing. The synonymous mixture index identified samples likely to harbor dual infection, while ultra-deep sequencing captured more intra-host viral diversity than single genome sequencing at approximately 40% of the cost and 20% of the laboratory and analysis time. The synonymous mixture index and ultra-deep sequencing are promising methods for rapid and cost-effective systematic identification of HIV dual infection.
Asunto(s)
Variación Genética , Infecciones por VIH/virología , VIH/clasificación , VIH/aislamiento & purificación , Virología/métodos , Adulto , Análisis por Conglomerados , VIH/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
BACKGROUND: Most of our knowledge about how antiretrovirals and host immune responses influence the HIV-1 protease gene is derived from studies of subtype B virus. We investigated the effect of protease resistance-associated mutations (PRAMs) and population-based HLA haplotype frequencies on polymorphisms found in CRF01_AE pro. METHODS: We used all CRF01_AE protease sequences retrieved from the LANL database and obtained regional HLA frequencies from the dbMHC database. Polymorphisms and major PRAMs in the sequences were identified using the Stanford Resistance Database, and we performed phylogenetic and selection analyses using HyPhy. HLA binding affinities were estimated using the Immune Epitope Database and Analysis. RESULTS: Overall, 99% of CRF01_AE sequences had at least 1 polymorphism and 10% had at least 1 major PRAM. Three polymorphisms (L10 V, K20RMI and I62 V) were associated with the presence of a major PRAM (P < 0.05). Compared to the subtype B consensus, six additional polymorphisms (I13 V, E35D, M36I, R41K, H69K, L89M) were identified in the CRF01_AE consensus; all but L89M were located within epitopes recognized by HLA class I alleles. Of the predominant HLA haplotypes in the Asian regions of CRF01_AE origin, 80% were positively associated with the observed polymorphisms, and estimated HLA binding affinity was estimated to decrease 19-40 fold with the observed polymorphisms at positions 35, 36 and 41. CONCLUSION: Polymorphisms in CRF01_AE protease gene were common, and polymorphisms at residues 10, 20 and 62 most likely represent selection by use of protease inhibitors, whereas R41K and H69K were more likely attributable to recognition of epitopes by the HLA haplotypes of the host population.
Asunto(s)
Antirretrovirales/uso terapéutico , Farmacorresistencia Viral/genética , Infecciones por VIH/virología , Proteasa del VIH/genética , VIH-1/genética , Polimorfismo Genético , Secuencia de Aminoácidos/genética , Pueblo Asiatico , Farmacorresistencia Viral/inmunología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/clasificación , VIH-1/efectos de los fármacos , VIH-1/enzimología , Humanos , Mutación/genéticaRESUMEN
Typically, population-based sequencing of HIV does not detect minority variants present at levels below 20-30%. Single genome amplification (SGA) and sequencing improves detection, but it requires many PCRs to find the optimal terminal dilution to use. A novel method for guiding the selection of a terminal dilution was developed and compared to standard methods. A quantitative real-time PCR (qRT-PCR) protocol was developed. HIV RNA was extracted, reverse transcribed, and quantitated. A bioinformatics web-based application was created for calculating the optimal concentration of cDNA to use based on results of a trial PCR using the dilution suggested by the qRT-PCR results. This method was compared to the standard. Using the standard protocol, the mean number of PCRs giving an average of 30 (26-34, SD=3) SGA per sample was 245 (218-266, SD=20) after an average of 8 trial dilutions. Using this method, 135 PCRs (135-135, SD=0) produced 30 (27-30, SD=1) SGA using exactly two dilutions. This new method reduced turnaround time from 8 to 2 days. Standard methods of SGA sequencing can be costly and both time- and labor-intensive. By choosing a terminal dilution concentration with the proposed method, the number of PCRs required is decreased and efficiency improved.
Asunto(s)
Biología Computacional/métodos , ADN Complementario/análisis , Genoma Viral/genética , VIH-1/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/aislamiento & purificación , Análisis de Secuencia de ADN/métodos , ADN Complementario/genética , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Distribución de Poisson , ARN Viral/sangre , ARN Viral/genética , ARN Viral/metabolismo , Carga ViralRESUMEN
BACKGROUND: Current public health efforts often use molecular technologies to identify and contain communicable disease networks, but not for HIV. Here, we investigate how molecular epidemiology can be used to identify highly related HIV networks within a population and how voluntary contact tracing of sexual partners can be used to selectively target these networks. METHODS: We evaluated the use of HIV-1 pol sequences obtained from participants of a community-recruited cohort (n = 268) and a primary infection research cohort (n = 369) to define highly related transmission clusters and the use of contact tracing to link other individuals (n = 36) within these clusters. The presence of transmitted drug resistance was interpreted from the pol sequences (Calibrated Population Resistance v3.0). RESULTS: Phylogenetic clustering was conservatively defined when the genetic distance between any two pol sequences was less than 1%, which identified 34 distinct transmission clusters within the combined community-recruited and primary infection research cohorts containing 160 individuals. Although sequences from the epidemiologically linked partners represented approximately 5% of the total sequences, they clustered with 60% of the sequences that clustered from the combined cohorts (odds ratio 21.7; P < or = 0.01). Major resistance to at least one class of antiretroviral medication was found in 19% of clustering sequences. CONCLUSION: Phylogenetic methods can be used to identify individuals who are within highly related transmission groups, and contact tracing of epidemiologically linked partners of recently infected individuals can be used to link into previously defined transmission groups. These methods could be used to implement selectively targeted prevention interventions.
Asunto(s)
Trazado de Contacto/métodos , Infecciones por VIH/transmisión , VIH-1/genética , Adulto , California/epidemiología , Farmacorresistencia Viral , Métodos Epidemiológicos , Femenino , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/efectos de los fármacos , VIH-1/aislamiento & purificación , Humanos , Masculino , Modelos Organizacionales , Filogenia , Parejas Sexuales , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
We assessed the effect of herpes simplex virus type 2 (HSV-2) acquisition on the plasma HIV RNA and CD4 cell levels among individuals with primary HIV infection using a retrospective cohort analysis. We studied 119 adult, antiretroviral-naive, recently HIV-infected men with a negative HSV-2-specific enzyme immunoassay (EIA) result at enrollment. HSV-2 acquisition was determined by seroconversion on HSV-2 EIA, confirmed by Western blot analysis. Ten men acquired HSV-2 infection a median of 1.3 years after HIV infection (HSV-2 incidence rate of 7.4 per 100 person-years of follow-up). The median time of follow-up after acquiring HSV-2 infection was 303 days. All men except 1 were asymptomatic during HSV-2 acquisition, and only 1 HSV-2 seroconverter, who was asymptomatic, had a transient increase in blood HIV load (0.5 log10 copies/mL over 11 days). The HSV-2 incidence rate was high in our cohort of recently HIV-infected individuals; however, HSV-2 acquisition did not significantly change the plasma HIV dynamics and CD4 cell levels.
Asunto(s)
Infecciones por VIH/complicaciones , VIH/aislamiento & purificación , Herpes Simple/complicaciones , Herpesvirus Humano 2 , ARN Viral/sangre , Adulto , Recuento de Linfocito CD4 , Estudios de Cohortes , Infecciones por VIH/virología , Herpes Simple/virología , Herpesvirus Humano 2/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Carga ViralRESUMEN
Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.
Asunto(s)
Canales de Sodio/genética , Canales de Sodio/fisiología , Secuencia de Aminoácidos , Animales , Evolución Molecular , Datos de Secuencia Molecular , Filogenia , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Alineación de SecuenciaRESUMEN
Many channels and carriers associate with auxiliary subunits which modify their activities and facilitate biogenesis. Advances in genome sequencing as well as biochemical, molecular genetic, and physiological experimentation have allowed for the discovery of many transport auxiliary subunits. Recent interests in the pharmacology of the calcium auxiliary subunits prompted a large amount of effort in deciphering their specific role in the conductance of calcium ions. In this review, we evaluate the functions of the 'extra' subunits of the voltage-gated calcium channels in animals as an example of auxiliary subunits of transporters in general. We discuss the functional data available for each of these subunits, present phylogenetic analyses, and discuss their potential evolutionary origins. Our analyses also reveal novel homologues of these subunits which might be of interest to the community.